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Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis
Thrombosis Research 125 (2010) 72–78
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis☆,☆☆ April P. McDonald a, Thomas R. Meier a,b,1, Angela E. Hawley a, Jacklyn N. Thibert a, Diana M. Farris a, Shirley K. Wrobleski a, Peter K. Henke a, Thomas W. Wakefield a, Daniel D. Myers Jr. a,b,⁎ a b
Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, USA Unit for Laboratory Animal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, USA
a r t i c l e
i n f o
Article history: Received 15 January 2009 Received in revised form 30 April 2009 Accepted 22 June 2009 Available online 18 July 2009 Keywords: Thrombus resolution Venous thrombosis Aging P-selectin PAI-1 Microparticles Tissue factor activity Animal models
a b s t r a c t Introduction: To evaluate the effects of aging on venous thrombosis. Material and Methods: Anesthetized male mice (C57BL/6, n = 125) underwent complete inferior vena cava occlusion to produce venous thrombosis. Experimental groups included 11-month-old mice (OLD), 2-monthold mice (YOUNG), and age-matched non-thrombosed controls. Mice were euthanized and the following parameters were evaluated two days post-thrombosis: thrombus mass (grams/cm), vein wall inflammatory cells (cells per 5 high powered fields), active plasma plasminogen activator inhibitor-1 (PAI-1, ng/mL), vein wall P-selectin protein determination by ELISA (pg/mL), circulating plasma microparticles (MPs, MPs/ 200 µL), MP tissue factor (TF) activity (pM), and in vivo MP re-injection experiments. Results: Thrombosed OLD mice had greater thrombus mass than YOUNG mice (389 ± 18 vs. 336 ± 14 g×10− 4/cm, P b.05). OLD mice had decreased vein wall monocyte, lymphocyte, and total inflammatory cell populations versus YOUNG mice (P b.05). Vein wall P-selectin levels were greater in OLD thrombosed mice versus YOUNG (7306± 938 vs. 3805 ± 745 pg/mL, P b.05). Active plasma PAI-1 concentrations were increased in OLD mice versus YOUNG thrombosed animals (20±4 vs. 8±2 ng/mL, P b.05). OLD mice had significantly higher circulating leukocytederived MPs versus YOUNG mice (5817± 850 vs. 2563± 283 MPs/200 μL PPP, P b.01). OLD mice had plasma MPs with increased TF activity versus YOUNG animals post-thrombosis (34± 4 vs. 24± 2 pM, Pb.05). Finally, YOUNG recipient animals, whether re-injected with OLD or YOUNG donor MPs, had a significant increase in thrombus mass versus OLD recipient animals (Pb.01). Conclusion: Aging influenced several circulating and vein wall factors that decreased thrombus resolution in older animals compared to younger ones in our mouse thrombosis model. © 2009 Elsevier Ltd. All rights reserved.
Introduction Venous thromboembolism (VTE), the collective term for deep vein thrombosis (DVT) and pulmonary embolism (PE), is a considerable healthcare concern in the United States, especially among the elderly [1–4]. The incidence of VTE increases markedly with advancing age.
☆ This original work was partially presented as an oral presentation at the American Venous Forum 20th Annual Meeting, Charleston, South Carolina. February 21, 2008. ☆☆ Financial Support: Diversity & Career Development Office (DCDO) [to DDM], Esperance Family Foundation (to APM), and Department of Surgery University of Michigan (to DDM). ⁎ Corresponding author. Section of Vascular Surgery/ Unit for Laboratory Animal Medicine (ULAM), Director, Jobst Vascular Research Laboratories, University of Michigan Medical School, A570D, MSRB II, 1150 W. Medical Center Drive, Dock#6, Ann Arbor, MI 48109-0654. Tel.: +1 734 763 0940; fax: +1 734 763 7307. E-mail address: [email protected] (D.D. Myers). 1 Dr. Thomas R. Meier is currently affiliated with Mayo Clinic, Rochester, MN, USA. 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.06.005
Approximately 450,000 incident VTE cases are reported each year, conservatively [4,5]. Of those, an estimated 187,000 cases are firsttime VTEs among those aged 45 years or older [1]. The incidence of VTE is 2-7 times higher in those greater than 55 years of age as compared to younger aged groups, and the incidence increases 74% per decade of age over 45 years [4]. Nearly half of cases diagnosed as thrombotic are idiopathic, and the rate of recurrence increases with age [1,2]. It is likely that the dramatic increase in VTE incidence with advancing age is reflective of the biology of aging [1–3]. Molecular and anatomic changes within the vessel wall, in addition to hemostatic alterations, may contribute to the susceptibility of thrombosis in the aging population. An important molecule implicated in age-associated thrombosis in the cardiovascular system is plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of fibrinolysis. Both lysed endothelial cells and platelets, activated due to injury, increase the synthesis of PAI-1 [6]. The expression of PAI-1 is elevated in the aged person and is greatly influenced by a multitude of pathologies associated with the process of aging, including obesity, insulin resistance, and vascular remodeling [2]. Ultimately, the
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presence of increased levels of PAI-1 in vivo may impair the normal fibrinolytic system creating a greater proclivity toward thrombosis. P-selectin and microparticles (MPs) are other molecules that have been identified in the pathogenesis of both arterial and venous thrombosis (VT) [7–12]. Selectins are expressed by activated platelets and endothelial cells and are the first glycoproteins up-regulated during inflammation [13]. The interaction of P-selectin with its receptor, P-selectin glycoprotein ligand-1, promotes a prothrombotic environment and the release of MPs, small fragments of cell membranes shed from platelets, leukocytes, and endothelial cells that are tissue factor-rich [13,14]. Microparticles promote and amplify coagulation once recruited to an area of thrombosis [9–11,15]. Specifically, leukocyte microparticles have been suggested to play a key role in VT. Clearly, there is a vast interplay among several procoagulant/antifibrinolytic factors of which the aberrant expression of any combination of them may underlie the increase in the incidence of VT observed in the elderly. Yet, the full identification of these molecules and the extent of their expression in an aged population have yet to be elucidated. The aim of this study was to identify molecular factors associated with aging, as compared to young, in VT resolution. We hypothesized that aged animals would have increased venous thrombus formation due to pro-inflammatory and impaired lytic factors. Materials and Methods Mouse Inferior Vena Cava Stasis Model The study utilized two populations of male C57BL/6 (wild type) mice for a total of 129 animals. Experimental groups included 2-month-old mice (YOUNG) (Harlan Sprague Dawley, Inc., Indianapolis, IN), 11-month-old mice (OLD) (Harlan Sprague Dawley, Inc.), and naïve non-thrombosed controls from the same background. Mice weighing 20-25 g (YOUNG) and 29-40 g (OLD) were anesthetized with an inhalation mixture of isoflurane gas (1.5 to 2%) and oxygen (100%) for the duration of the procedure. A midline laparotomy was made, the small bowel was exteriorized, and the inferior vena cava (IVC) was approached directly via blunt dissection. Just inferior to the renal veins the IVC and associated side branches were ligated with 7-0 prolene suture. Large dorsal branches were cauterized. The laparotomy was closed in a two-layer fashion using 5-0 vicryl and Vetbond tissue adhesive (3 M Animal Care Products, St. Paul, MN) [12,16]. Mice were euthanized two days post-thrombosis for tissue harvest and blood collection. Thrombus Mass At the time of euthanasia, the IVC and associated thrombus was removed, weighed (grams), and measured for length (cm). The weight was then normalized to length (weight/length measurement) [16]. Vein Wall Morphometrics Tissue sections were fixed and embedded in paraffin using standard histological methods and subsequently stained with hematoxylin and eosin [16]. Five representative high-power fields were examined around the vein wall (oil immersion light microscopy, 1000x) and the cell count of the vein wall was analyzed in a blinded fashion. Cells were identified as neutrophils, monocyte/macrophages, or lymphocytes based on standard histological criteria including nuclear size, cytoplasm content, and total cell size as previously described [16]. From the sums of the five high-power fields the mean± SE was calculated for each vein section studied.
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Sandwich ELISA: Vein Wall P-selectin Protein Quantification Vein wall tissue sections were evaluated for P-selectin using a sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) [Pierce Biotechnology, Woburn, MA]. In brief, all samples were run in duplicate on freshly homogenized vein wall tissue, with homogenization performed in complete protease inhibitor cocktail (Roche Mannheim, Germany). The suspension was sonicated on ice for 30 seconds, centrifuged at 14000 rpm for 10 minutes, and the supernatant used to run the tests. The tests were run according to the manufacturer's suggestions. The plates were read on a Plate Reader Elx808 (Biotek, Vermont) at 450 nm wavelength. Total protein is measured using a bicinchoninic acid (BCA) protein assay kit assessing colorimetric detection and quantitation of total protein, read at 590 nm (Pierce, Rockford, IL). Protein levels within each group were reported as mean ± SE pg/mL. Plasma plasminogen activator inhibitor-1 (PAI-1) Determination At the time of euthanasia, blood samples were collected by intracardiac puncture. Blood was drawn into a syringe containing 3.8% sodium citrate (9:1 v:v), placed in an eppendorf tube, and immediately centrifuged at 3,000 ×g for 15 minutes. Plasma was snap frozen in liquid nitrogen then stored at minus 70 degrees centigrade. On the morning of the determination, plasma was thawed and active plasma PAI-1 determined using a commercially available kit (Molecular Innovations RPAIKT, Southfield, MI) that utilized an Enzyme-Linked ImmunoSorbent Assay (ELISA). These data were evaluated (ng/mL) and the means ± SEM and p-values were subsequently calculated. Murine Platelet and Leukocyte Microparticle Analysis Whole blood (400 μL) was removed from mice by intracardiac puncture using a syringe primed with 10% acid citrate dextrose. Blood was placed in an eppendorf tube centrifuged at 1,500 ×g and 23 °C for 25 minutes, plasma transferred to an additional eppendorf tube and spun again for 2 minutes at 15,000 ×g to obtain platelet poor plasma (PPP). Two hundred micro liters (200 μL) of PPP obtained from each mouse was diluted 1:3 with 600 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Additionally, 100 μL of individual mouse plasma was pooled and divided into three equal volumes and added to 600 μL of HEPES buffer for internal controls. All samples were centrifuged for 2 hours at 200,000 ×g to separate the microparticles (MPs). The supernatant was removed, and pelleted MPs were resuspended in HEPES buffer. Rat anti-murine PE (Chemicon International, Temecula, CA), or control rat PE IgG (BD Pharmingen, San Diego, CA), and rat anti-murine fluorescein isothiocyanate (BD Pharmingen) or control rat FITC IgG (BD Pharmingen) were added to samples to stain leukocytes and platelets. Murine antibody controls consisted of the following: FITC/PE, 1.5 μL/ 0.6 μL (negative control), leukocyte marker MAC-1 (PE), 12.0 μL (positive control), and platelet marker FITC (CD41), 1.2 μL (positive control). For MP quantification, a known quantity (250,000) of 3.0 – 3.4 µm beads (SPHERO™, BD Pharmingen) was added to each fixed sample prior to FACS analysis, and acquisition was stopped after 50,000 beads were counted in the R-1 bead gate. The diameter of these beads allowed discrimination from the MP population (1.0 µm or less) on light scatter and were counted using a separate bead gate as previously described [12]. Circulating Tissue Factor Activity Two hundred microliters of platelet poor plasma (PPP) was obtained by centrifuging blood at 1,500 ×g for 25 minutes. The plasma was removed and centrifuged once more for 2 minutes at 15,000 ×g to remove contaminating cells from the plasma. The tissue factor assay
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Statistical Evaluation and Animal Use All analyses were performed using SigmaStat (v.2.03). The statistical differences between groups were determined by the Student's t-test. Non-parametric data was assessed using the MannWhitney Rank Sum. A value of P ≤ .05 was considered significant and data reported as mean ± SE. All mice were housed and cared for by the University of Michigan Unit for Laboratory Animal Medicine and were free of pathogens. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals [19]. The University of Michigan Committee on Use and Care of Animals approved this research protocol. Results Fig. 1. Thrombus mass was significantly increased in OLD mice compared to YOUNG animals 2 days post venous thrombosis. YOUNG non-thrombosed control animals had significantly larger venous thrombi than OLD mice.
kit (American Diagnostica, Stamford, CT) combined soluble factor VIIa, Human Factor X, TF/TFPI free plasma, and the MP sample (spun down as above, but the pellet was resuspended in TF/TFPI free plasma). This combination activated Factor X, which then hydrolyzed Spectrozyme Xa (American Diagnostica Inc., Greenwich, CT). This hydrolysis occurred in a linear fashion at 37 °C as read on a spectrophotometer. Enzyme activity was determined by measuring the increase in absorbance of the free chromophore (pNA) generated per unit time at λ405 nm. The positive control was a known amount of human tissue factor (standard curve) while the negative control was microparticle free buffer. Human reagents were used to evaluate mouse tissue factor activity for comparative data analysis only.
Aging Increases Thrombus Formation On day 2, post-thrombosis, all mice were euthanized and their vein wall and associated thrombus were weighed and normalized to the length of the affected vein wall segment to evaluate thrombus mass. OLD mice (n = 8) demonstrated significantly larger venous thrombi than YOUNG animals (n = 8) (389 ± 18 vs. 336 ± 14 g × 10− 4/cm, P b.05) [Fig. 1]. Older Mice Have Decreased Vein Wall Inflammatory Cells OLD mice (n= 8) had significantly fewer vein wall monocyte (4± 0.4 vs.12 ± 2 cells/HPFs, P b.01), lymphocyte (1± 0.1 vs. 5 ± 2 cells/HPFs, P b.05), and total inflammatory cell populations (16 ± 3 vs. 29 ± 5 cells/ HPFs, P b.05) than YOUNG mice (n= 8), 2 days post-thrombosis (Fig. 2). No significant differences existed between OLD and YOUNG nonthrombosed control animals (data not shown).
Microparticle Re-injection Experiments
Vein Wall Protein Determination
Donor MP Preparation Mice from both OLD (n = 23) and YOUNG (n = 21) experimental groups underwent IVC ligation to induce thrombosis, as previously described [16,17]. Two days post-thrombosis, mice had whole blood withdrawn for the plasma microparticle assay, as previously described [12]. Mice were then euthanized and their IVC and associated thrombus weighed. The platelet poor plasma (PPP) was pooled from donor animals within each respective group and thoroughly mixed into two separate heterogeneous stock solutions. These stock solutions were re-aliquoted in equal amounts, snap frozen in liquid nitrogen − 196 °C, and then stored at −70 °C. A single aliquot of donor PPP was analyzed by flow cytometry to determine the total MP concentration of 160,000 MPs/200 µL of PPP. Prior to injection into recipient mice, MP aliquots were thawed and centrifuged in a 1:3 ratio with HEPES buffer pH 7.4 at 4 °C for 2 hours at 200,000 ×g. The supernatant was removed and pelleted MPs were resuspended in 100 µL of a 10 mM HEPES, 136 mM NaCl, 5 mM MgCl2, 50 mM KCl (pH 7.4) buffer for injection into recipient animals.
Aging Increases Vein Wall P-selectin OLD non-thrombosed control mice showed a trend of elevated vein wall P-selectin versus YOUNG mice. Two days post thrombosis, OLD thrombosed mice (n= 8) showed a statistically significant increase in vein wall P-selectin levels compared to YOUNG animals (n= 8) [7306 ± 938 vs. 3805 ± 745 pg/mL, P b.05] (Fig. 3).
Recipient Animal Preparation Recipient mice from OLD (n = 20) and YOUNG (n = 12) groups were anesthetized with isoflurane gas. 100 µL of the heterogeneous MP solution (160,000 MPs/100 µL of PPP) was administered into the penile vein. This concentration of MPs re-injected into recipient mice consistently produces venous thrombosis comparable to control mice that have undergone complete IVC ligation [18]. After the MP injection, the mice were surgically prepped and underwent IVC ligation. Two days post IVC thrombosis, the mice were euthanized and their IVC and associated thrombus were removed, weighed and normalized to length (thrombus mass) as previously described [16].
Plasma Plasminogen Activator Inhibitor-1 (PAI-1) Concentration Increases with Age Active plasma PAI-1 was measured in samples from both nonthrombosed controls and animals 2 days post thrombosis. In mice with thrombus present for 2 days, active plasma PAI-1 concentrations
Fig. 2. OLD mice had significantly fewer monocytes, lymphocytes, and total inflammatory cell populations migrating through the vein wall versus YOUNG animals.
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0.8 grams × 10− 4/cm/animal weight, P b.01) [Fig. 6]. Therefore, young recipient animals receiving microparticles from either young or old donors showed significant increases in thrombus mass when indexed to animal weight two days post thrombosis.
Discussion
Fig. 3. OLD thrombosed mice showed a significant increase in vein wall P-selectin levels compared to YOUNG animals post-thrombosis.
were significantly increased in OLD mice (n = 6) versus YOUNG animals (n = 6) [20 ± 4 vs. 8 ± 2 ng/mL, P b.05] (Fig. 4). Circulating Leukocyte-derived Microparticles Increase with Age OLD thrombosed animals (n = 10) displayed significantly higher circulating leukocyte-derived microparticles (MAC-1) than YOUNG mice (n = 9) [5817 ± 850 vs. 2564 ± 283 MPs/200 µL PPP, P b.01]. Platelet-derived (CD41) and total microparticles were non-significantly increased in OLD mice compared to YOUNG animals (Fig. 5A). There were no baseline differences between OLD and YOUNG nonthrombosed control groups evaluated for plasma microparticles. Microparticle Tissue Factor Activity Increases in Older Mice Mice from both OLD and YOUNG experimental groups had blood withdrawn for plasma MP assays 2 days post venous thrombosis. Plasma MPs from each mouse were then assayed in duplicate for TF procoagulant activity. OLD mice (n = 17) had plasma MPs with significantly higher TF activity 2 days post-thrombosis when compared to YOUNG (n = 11) animals (34 ± 4 vs. 24 ± 2 pM, P b.05) [Fig. 5B]. Younger Mice Receiving Microparticles Have Increased Thrombotic Potential In order to determine the acute thrombotic potential of MPs, murine MPs from OLD and YOUNG donor pools were re-injected into age-matched recipient mice at the time of thrombus induction with thrombus size evaluated 2 days later. Recipient animals received a concentration of 160,000 MPs in 100 µL of PPP from the age-matched donor pool and then underwent IVC thrombosis. Two days post reinjection of donor MPs, mice were euthanized for sample analysis. Both donor and recipient animals had their thrombus mass divided by animal body weight (grams) to normalize for size differences between young and older mice. When MPs from OLD donor animals, 2 days post thrombosis, were re-injected into YOUNG recipient animals (OLD-D into YOUNG-R, n = 5), there was an increased thrombus burden versus OLD recipient animals receiving MPs from OLD donors (OLD-D into OLD-R, n = 11) or YOUNG donor mice (YOUNG-D into OLD-R, n = 9), 2 days post thrombosis, (17 ± 1 vs. 12 ± .0.4 grams ± 10− 4/cm/animal weight, P b.01,13 ± 0.8 grams × 10− 4/cm/animal weight, P b.05). Microparticles from YOUNG donor mice re-injected into YOUNG recipients (YOUNG-D into YOUNG-R, n = 7) had significantly larger venous thrombi when compared to older mice receiving MPs from OLD (OLD-D into OLD-R, n = 11) or YOUNG (YOUNG-D into OLD-R, n = 9) donor mice, 2 days post thrombosis (16 ± 0.7 vs.12 ± .0.4, 13 ±
Using a mouse model of VT, our data showed that aging was associated with increased thrombus size in mice. We reported that several prothrombotic circulating and vein wall factors were elevated in older animals when compared to young mice undergoing stasis induced thrombosis. These included: 1) decreased vein wall inflammatory cells; 2) increases plasma PAI-1; 3) increased vein wall P-selectin; 4) increased leukocyte MPs levels; 5) increased MP tissue factor activity. Importantly, the donor/recipient experiments suggest the host age and physiological environment are important factors in stasis thrombus resolution. Older mice had significantly greater thrombus mass when compared to younger animals. Consistently, OLD animals had significantly decreased vein wall inflammatory cell populations, which may affect thrombus resolution in these animals [20]. In a previous natural history study using the same IVC ligation model of venous thrombosis, we documented an early neutrophil (PMN) cell vein wall inflammatory cell influx followed by a later influx of monocytes during thrombosis [16]. Neutrophils and monocytes both have a defined role in inflammation and thrombus resolution. For example, in a rat IVC ligation model of venous thrombosis, neutrophil depletion of these animals promoted larger venous thrombi associated with increased thrombus fibrosis in animals evaluated at day 2 and day 7 [21]. PMNs are necessary for early thrombus resolution by promoting the lysis of both fibrin and collagen [22,23]. Inflammatory cells, especially monocytes, have been shown to promote thrombolysis due to their secretion of proteolytic enzymes [24–26]. Post thrombosis, increasing levels of monocyte chemotactic protein-1 (MCP-1) stimulate chemotaxis and activation of monocytes which promote venous thrombus resolution [27] . In this study, YOUNG animals with significantly greater vein wall inflammatory cell populations had significantly smaller venous thrombi. PAI-1 inhibits the serine proteases tPA and uPA, and thus fibrinolysis, the process which promotes thrombus resolution. OLD thrombosed animals had a significant increase in circulating PAI-1 activity compared to YOUNG. PAI-1 has been shown to significantly increase with age in both plasma and murine tissues and in aged rats, which supports our findings [28–30]. Recent clinical studies have reviewed the relationship between aging and increased circulating levels of PAI-1. With age, factors such as obesity and stress, promote increased production of PAI-1 levels [6,28,29,31,32]. In this study,
Fig. 4. Active plasma plasminogen activator inhibitor-1 (PAI-1) in OLD mice was significantly elevated at 2 days post thrombosis when directly compared to YOUNG thrombosed animals at the same time point.
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Fig. 5. A. OLD thrombosed animals displayed significantly higher circulating leukocyte-derived microparticles (MAC-1) than YOUNG mice. B: OLD mice had plasma microparticles with significantly higher tissue factor activity 2 days post-thrombosis versus YOUNG mice.
significantly increased PAI-1 activity in OLD mice suggests reduced fibrinolysis, which may explain the causal relationship with the larger thrombi observed in the OLD mice. Previous studies by our laboratory and others have shown that increases in P-selectin and interactions of P-selectin with its receptor, P-selectin glycoprotein-1 (PSGL-1), promote leukocyte rolling and
Fig. 6. To determine the thrombotic potential of microparticles (MPs), murine MPs from OLD and YOUNG donor pools of animals were re-injected into recipient mice of different ages 2 days post-thrombosis. Two days post re-injection of MPs into recipient mice; animals were humanely euthanized and had their thrombus mass evaluated. A significant increase in thrombus burden in YOUNG recipient animals was noted.
emigration at the site of injured vascular endothelium encouraging thrombogenesis [7,10,33]. Using rodent models of venous thrombosis to evaluate P-selectin inhibition, we have documented that thrombosis can be inhibited without total inhibition of vein wall inflammatory cells [17,34]. These data suggest that other inflammatory mechanisms, in addition to P-selectin promote inflammatory cell extravasation through the vein wall. Thus, the amount of P-selectin and vein wall inflammatory cells does not correlate. This lack of correlation was evident in a study testing the effects of a small molecule inhibitor of Pselectin on venous thrombosis. In this rodent study, P-selectin inhibition effectively decreased vein wall fibrosis without decreasing vein wall inflammatory cells extravasation [34]. Another contributor to the increased procoagulant state of the OLD mice may be the significantly increased levels of circulating plasma leukocyte-derived MPs. With damage to the venous endothelium, Pselectin becomes expressed on endothelial cells and platelets simulating interactions between P-selectin and its receptor, PSGL-1. P-selectin: PSGL-1 interactions produce the generation of MPs from leukocytes post vascular injury [7,8,10,12]. Such interactions activate procoagulant MP release from platelets, leukocytes and endothelial cells. Tissue factor is primarily responsible for thrombosis. Work by Nemerson determined that TF is naturally circulating on a subpopulation of microparticles [35]. These TF-bearing MPs have been shown to accumulate in the developing thrombus after vascular injury [15,28,36]. This mechanism is driven by P-selectin: PSGL-1 interactions [9,10,36]. In the current study, mice had plasma MPs evaluated for circulating TF activity 2 days post venous thrombogenesis. OLD animals had significantly higher concentrations of MP TF activity than YOUNG animals. These findings suggest that leukocyte MPs have increased TF activity, and subsequently increases thrombosis.
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Although our laboratory and others have consistently been able to detect mouse plasma tissue factor activity using human reagents, we recognize that mouse TF may not have the same specificity or interactions as human TF. It has been shown that human TF interacts effectively with both human and mouse VIIa [37,38]. However the converse is not true, mouse TF interacts poorly with human VIIa [37,38]. Therefore, it is possible that the true mouse plasma TF activity in this study was underreported in our OLD and YOUNG mice 2 days post thrombosis. Presently, multiple leaders in the field of tissue factor biology are working to develop specific murine tissue factor antibodies in an effort to standardize murine in vitro and in vivo TF assays. Future studies will also need to evaluate the role of latent/ encrypted TF during acute venous thrombosis. To determine if circulating MPs or the age of the recipient determines the thrombogenic response, we re-injected MPs obtained from donor animals 2 days post-thrombosis into age-matched recipient mice. Previous work in our laboratory has determined that acute inflammatory responses (increased vein wall inflammatory cell extravasation and P-selectin protein levels) are peaking at 2 days post venous thrombosis in our mouse model [16,17]. Thus, the 2 day post thrombosis time-point has documented pro-inflammatory activity present. YOUNG recipient animals had significantly larger venous thrombi 2 days post-injection of MPs regardless if the re-injected MPS were from YOUNG or OLD donor animals. This finding did not hold true for OLD recipient mice receiving donor MPs from OLD or YOUNG mice. In previous studies, mice with TFpositive MPs from mice with high circulating levels of P-selectin, showed that these MPs were incorporated into developing thrombi [11]. From these data, it can be hypothesized that MPs are playing a role in the development of large thrombi in YOUNG animals. This was an unexpected finding in this study. Based on the other factors identified in this study, we hypothesized the MPs re-injected into older animals with impaired mechanisms of thrombolysis (significantly elevated circulating active PAI-1), should have larger venous thrombi. It is clear that other mechanisms are at play such as host environment (blood and endothelium) and duration of thrombosis. Mice were evaluated 2 days post thrombosis which is an early time point for this dynamic process. Future experiments warrant evaluating both early and late thrombotic events in animals at different ages to determine the true mechanism behind these results. The phenotype of YOUNG animals appears to promote the development of thrombi of the same magnitude in these re-injection experiments. This suggests that the recipient's physiological environment is more important than the age of the donor of MPs. We did not formally evaluate coagulation tests in this study however, there were no adverse effects such as wound healing or bleeding complications detected in either OLD or YOUNG mice. In conclusion, we have shown differences in several circulating and vein wall factors that influence thrombosis between OLD and YOUNG animals in our rodent model. We hypothesize that with increasing age changes occur in both the venous endothelium and circulation that promote a prothrombotic environment. These factors include increased activity in PAI-1 and procoagulant MPs, a sub-population that has measured TF activity. In addition, there are active mechanisms that acutely modulate vein wall inflammatory cell populations while favoring P-selectin expression producing a thrombogenic vascular environment in OLD animals. These data collectively suggest possible mechanisms influencing the increase in VT that occurs with aging. Conflicts of interest The authors of this manuscript have no conflicts of interest to disclose. Acknowledgements Financial support was obtained from the Diversity & Career Development Office (DCDO) [to DDM], Esperance Family Foundation
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(to APM), and Department of Surgery University of Michigan (to DDM). We thank Ms. Iris Richardson for her technical assistance. References [1] Cushman M, Tsai AW, White RH, Heckbert SR, Rosamond WD, Enright P, et al. Deep vein thrombosis and pulmonary embolism in two cohorts: the longitudinal investigation of thromboembolism etiology. Am J Med 2004;117:19–25. [2] Heit JA. Venous thromboembolism: disease burden, outcomes and risk factors. J Thromb Haemost 2005;3:1611–7. [3] Silverstein MD, Heit JA, Mohr DN, Petterson TM, O'Fallon WM, Melton III LJ. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Arch Intern Med 1998;158:585–93. [4] Tsai AW, Cushman M, Rosamond WD, Heckbert SR, Polak JF, Folsom AR. Cardiovascular risk factors and venous thromboembolism incidence: the longitudinal investigation of thromboembolism etiology. Arch Intern Med 2002;162:1182–9. [5] Goldhaber SZ. Pulmonary embolism. Lancet 2004;363:1295–305. [6] Yamamoto K, Takeshita K, Kojima T, Takamatsu J, Saito H. Aging and plasminogen activator inhibitor-1 (PAI-1) regulation: implication in the pathogenesis of thrombotic disorders in the elderly. Cardiovasc Res 2005;66:276–85. [7] Andre P, Hartwell D, Hrachovinova I, Saffaripour S, Wagner DD. Pro-coagulant state resulting from high levels of soluble P-selectin in blood. Proc Natl Acad Sci U S A 2000;97:13835–40. [8] Cambien B, Wagner DD. A new role in hemostasis for the adhesion receptor P-selectin. Trends Mol Med 2004;10:179–86. [9] Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, et al. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle Pselectin glycoprotein ligand 1 and platelet P-selectin. J Exp Med 2003;197:1585–98. [10] Furie B, Furie BC. Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation. Trends Mol Med 2004;10:171–8. [11] Hrachovinova I, Cambien B, Hafezi-Moghadam A, Kappelmayer J, Camphausen RT, Widom A, et al. Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A. Nat Med 2003;9:1020–5. [12] Myers DD, Hawley AE, Farris DM, Wrobleski SK, Thanaporn P, Schaub RG, et al. Pselectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg 2003;38:1075–89. [13] Myers Jr DD, Wrobleski SK, Longo C, Bedard PW, Kaila N, Shaw GD, et al. Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation. Thromb Haemost 2007;97:400–7. [14] Rectenwald JE, Myers Jr DD, Hawley AE, Longo C, Henke PK, Guire KE, et al. Ddimer, P-selectin, and microparticles: novel markers to predict deep venous thrombosis. A pilot study. Thromb Haemost 2005;94:1312–7. [15] Giesen PL, Rauch U, Bohrmann B, Kling D, Roque M, Fallon JT, et al. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci U S A 1999;96:2311–5. [16] Myers Jr D, Farris D, Hawley A, Wrobleski S, Chapman A, Stoolman L, et al. Selectins influence thrombosis in a mouse model of experimental deep venous thrombosis. J Surg Res 2002;108:212–21. [17] Myers Jr DD, Rectenwald JE, Bedard PW, Kaila N, Shaw GD, Schaub RG, et al. Decreased venous thrombosis with an oral inhibitor of P selectin. J Vasc Surg 2005;42:329–36. [18] Ramacciotti E, Hawley AE, Farris DM, Ballard NE, Wrobleski SK, Myers Jr DD, et al. Leukocyte- and platelet-derived microparticles correlate with thrombus weight and tissue factor activity in an experimental mouse model of venous thrombosis. Thromb Haemost 2009;101:748–54. [19] Institute of Laboratory Animal Resources CoLS, National Research Council. Guide for the care and use of laboratory animals. 7th ed. Washington, D.C.: National Academy Press; 1996. [20] Henke PK, Varma MR, Deatrick KB, Dewyer NA, Lynch EM, Moore AJ, et al. Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization. Thromb Haemost 2006;95:272–81. [21] Varma MR, Moaveni DM, Dewyer NA, Varga AJ, Deatrick KB, Kunkel SL, et al. Deep vein thrombosis resolution is not accelerated with increased neovascularization. J Vasc Surg 2004;40:536–42. [22] Stewart GJ. Neutrophils and deep venous thrombosis. Haemostasis 1993;23 (Suppl 1):127–40. [23] Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, et al. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg 2003;38:1090–8. [24] Burnand KG, Gaffney PJ, McGuinness CL, Humphries J, Quarmby JW, Smith A. The role of the monocyte in the generation and dissolution of arterial and venous thrombi. Cardiovasc Surg 1998;6:119–25. [25] Henke PK, Varga A, De S, Deatrick CB, Eliason J, Arenberg DA, et al. Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol 2004;24:1130–7. [26] Humphries J, McGuinness CL, Smith A, Waltham M, Poston R, Burnand KG. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg 1999;30:894–9. [27] Hogaboam CM, Steinhauser ML, Chensue SW, Kunkel SL. Novel roles for chemokines and fibroblasts in interstitial fibrosis. Kidney Int 1998;54:2152–9. [28] Chou J, Mackman N, Merrill-Skoloff G, Pedersen B, Furie BC, Furie B. Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation. Blood 2004;104:3190–7. [29] Hashimoto Y, Kobayashi A, Yamazaki N, Sugawara Y, Takada Y, Takada A. Relationship between age and plasma t-PA, PA-inhibitor, and PA activity. Thromb Res 1987;46:625–33. [30] Takeshita K, Yamamoto K, Ito M, Kondo T, Matsushita T, Hirai M, et al. Increased expression of plasminogen activator inhibitor-1 with fibrin deposition in a murine model of aging, "Klotho" mouse. Semin Thromb Hemost 2002;28:545–54.
78
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[31] Mari D, Coppola R, Provenzano R. Hemostasis factors and aging. Exp Gerontol 2008;43:66–73. [32] Yamamoto K, Takeshita K, Shimokawa T, Yi H, Isobe K, Loskutoff DJ, et al. Plasminogen activator inhibitor-1 is a major stress-regulated gene: implications for stress-induced thrombosis in aged individuals. Proc Natl Acad Sci U S A 2002;99:890–5. [33] Myers DD, Wakefield TW. Inflammation-dependent thrombosis. Front Biosci 2005;10:2750–7. [34] Myers Jr DD, Henke PK, Bedard PW, Wrobleski SK, Kaila N, Shaw G, et al. Treatment with an oral small molecule inhibitor of P selectin (PSI-697) decreases vein wall injury in a rat stenosis model of venous thrombosis. J Vasc Surg 2006;44:625–32.
[35] Nemerson Y. Tissue factor and hemostasis. Blood 1988;71:1–8. [36] Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 2002;8:1175–81. [37] Fang CH, Lin TC, Guha A, Nemerson Y, Konigsberg WH. Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3. Thromb Haemost 1996;76:361–8. [38] Janson TL, Stormorken H, Prydz H. Species specificity of tissue thromboplastin. Haemostasis 1984;14:440–4.
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis☆,☆☆ April P. McDonald a, Thomas R. Meier a,b,1, Angela E. Hawley a, Jacklyn N. Thibert a, Diana M. Farris a, Shirley K. Wrobleski a, Peter K. Henke a, Thomas W. Wakefield a, Daniel D. Myers Jr. a,b,⁎ a b
Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, USA Unit for Laboratory Animal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, USA
a r t i c l e
i n f o
Article history: Received 15 January 2009 Received in revised form 30 April 2009 Accepted 22 June 2009 Available online 18 July 2009 Keywords: Thrombus resolution Venous thrombosis Aging P-selectin PAI-1 Microparticles Tissue factor activity Animal models
a b s t r a c t Introduction: To evaluate the effects of aging on venous thrombosis. Material and Methods: Anesthetized male mice (C57BL/6, n = 125) underwent complete inferior vena cava occlusion to produce venous thrombosis. Experimental groups included 11-month-old mice (OLD), 2-monthold mice (YOUNG), and age-matched non-thrombosed controls. Mice were euthanized and the following parameters were evaluated two days post-thrombosis: thrombus mass (grams/cm), vein wall inflammatory cells (cells per 5 high powered fields), active plasma plasminogen activator inhibitor-1 (PAI-1, ng/mL), vein wall P-selectin protein determination by ELISA (pg/mL), circulating plasma microparticles (MPs, MPs/ 200 µL), MP tissue factor (TF) activity (pM), and in vivo MP re-injection experiments. Results: Thrombosed OLD mice had greater thrombus mass than YOUNG mice (389 ± 18 vs. 336 ± 14 g×10− 4/cm, P b.05). OLD mice had decreased vein wall monocyte, lymphocyte, and total inflammatory cell populations versus YOUNG mice (P b.05). Vein wall P-selectin levels were greater in OLD thrombosed mice versus YOUNG (7306± 938 vs. 3805 ± 745 pg/mL, P b.05). Active plasma PAI-1 concentrations were increased in OLD mice versus YOUNG thrombosed animals (20±4 vs. 8±2 ng/mL, P b.05). OLD mice had significantly higher circulating leukocytederived MPs versus YOUNG mice (5817± 850 vs. 2563± 283 MPs/200 μL PPP, P b.01). OLD mice had plasma MPs with increased TF activity versus YOUNG animals post-thrombosis (34± 4 vs. 24± 2 pM, Pb.05). Finally, YOUNG recipient animals, whether re-injected with OLD or YOUNG donor MPs, had a significant increase in thrombus mass versus OLD recipient animals (Pb.01). Conclusion: Aging influenced several circulating and vein wall factors that decreased thrombus resolution in older animals compared to younger ones in our mouse thrombosis model. © 2009 Elsevier Ltd. All rights reserved.
Introduction Venous thromboembolism (VTE), the collective term for deep vein thrombosis (DVT) and pulmonary embolism (PE), is a considerable healthcare concern in the United States, especially among the elderly [1–4]. The incidence of VTE increases markedly with advancing age.
☆ This original work was partially presented as an oral presentation at the American Venous Forum 20th Annual Meeting, Charleston, South Carolina. February 21, 2008. ☆☆ Financial Support: Diversity & Career Development Office (DCDO) [to DDM], Esperance Family Foundation (to APM), and Department of Surgery University of Michigan (to DDM). ⁎ Corresponding author. Section of Vascular Surgery/ Unit for Laboratory Animal Medicine (ULAM), Director, Jobst Vascular Research Laboratories, University of Michigan Medical School, A570D, MSRB II, 1150 W. Medical Center Drive, Dock#6, Ann Arbor, MI 48109-0654. Tel.: +1 734 763 0940; fax: +1 734 763 7307. E-mail address: [email protected] (D.D. Myers). 1 Dr. Thomas R. Meier is currently affiliated with Mayo Clinic, Rochester, MN, USA. 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.06.005
Approximately 450,000 incident VTE cases are reported each year, conservatively [4,5]. Of those, an estimated 187,000 cases are firsttime VTEs among those aged 45 years or older [1]. The incidence of VTE is 2-7 times higher in those greater than 55 years of age as compared to younger aged groups, and the incidence increases 74% per decade of age over 45 years [4]. Nearly half of cases diagnosed as thrombotic are idiopathic, and the rate of recurrence increases with age [1,2]. It is likely that the dramatic increase in VTE incidence with advancing age is reflective of the biology of aging [1–3]. Molecular and anatomic changes within the vessel wall, in addition to hemostatic alterations, may contribute to the susceptibility of thrombosis in the aging population. An important molecule implicated in age-associated thrombosis in the cardiovascular system is plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of fibrinolysis. Both lysed endothelial cells and platelets, activated due to injury, increase the synthesis of PAI-1 [6]. The expression of PAI-1 is elevated in the aged person and is greatly influenced by a multitude of pathologies associated with the process of aging, including obesity, insulin resistance, and vascular remodeling [2]. Ultimately, the
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presence of increased levels of PAI-1 in vivo may impair the normal fibrinolytic system creating a greater proclivity toward thrombosis. P-selectin and microparticles (MPs) are other molecules that have been identified in the pathogenesis of both arterial and venous thrombosis (VT) [7–12]. Selectins are expressed by activated platelets and endothelial cells and are the first glycoproteins up-regulated during inflammation [13]. The interaction of P-selectin with its receptor, P-selectin glycoprotein ligand-1, promotes a prothrombotic environment and the release of MPs, small fragments of cell membranes shed from platelets, leukocytes, and endothelial cells that are tissue factor-rich [13,14]. Microparticles promote and amplify coagulation once recruited to an area of thrombosis [9–11,15]. Specifically, leukocyte microparticles have been suggested to play a key role in VT. Clearly, there is a vast interplay among several procoagulant/antifibrinolytic factors of which the aberrant expression of any combination of them may underlie the increase in the incidence of VT observed in the elderly. Yet, the full identification of these molecules and the extent of their expression in an aged population have yet to be elucidated. The aim of this study was to identify molecular factors associated with aging, as compared to young, in VT resolution. We hypothesized that aged animals would have increased venous thrombus formation due to pro-inflammatory and impaired lytic factors. Materials and Methods Mouse Inferior Vena Cava Stasis Model The study utilized two populations of male C57BL/6 (wild type) mice for a total of 129 animals. Experimental groups included 2-month-old mice (YOUNG) (Harlan Sprague Dawley, Inc., Indianapolis, IN), 11-month-old mice (OLD) (Harlan Sprague Dawley, Inc.), and naïve non-thrombosed controls from the same background. Mice weighing 20-25 g (YOUNG) and 29-40 g (OLD) were anesthetized with an inhalation mixture of isoflurane gas (1.5 to 2%) and oxygen (100%) for the duration of the procedure. A midline laparotomy was made, the small bowel was exteriorized, and the inferior vena cava (IVC) was approached directly via blunt dissection. Just inferior to the renal veins the IVC and associated side branches were ligated with 7-0 prolene suture. Large dorsal branches were cauterized. The laparotomy was closed in a two-layer fashion using 5-0 vicryl and Vetbond tissue adhesive (3 M Animal Care Products, St. Paul, MN) [12,16]. Mice were euthanized two days post-thrombosis for tissue harvest and blood collection. Thrombus Mass At the time of euthanasia, the IVC and associated thrombus was removed, weighed (grams), and measured for length (cm). The weight was then normalized to length (weight/length measurement) [16]. Vein Wall Morphometrics Tissue sections were fixed and embedded in paraffin using standard histological methods and subsequently stained with hematoxylin and eosin [16]. Five representative high-power fields were examined around the vein wall (oil immersion light microscopy, 1000x) and the cell count of the vein wall was analyzed in a blinded fashion. Cells were identified as neutrophils, monocyte/macrophages, or lymphocytes based on standard histological criteria including nuclear size, cytoplasm content, and total cell size as previously described [16]. From the sums of the five high-power fields the mean± SE was calculated for each vein section studied.
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Sandwich ELISA: Vein Wall P-selectin Protein Quantification Vein wall tissue sections were evaluated for P-selectin using a sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) [Pierce Biotechnology, Woburn, MA]. In brief, all samples were run in duplicate on freshly homogenized vein wall tissue, with homogenization performed in complete protease inhibitor cocktail (Roche Mannheim, Germany). The suspension was sonicated on ice for 30 seconds, centrifuged at 14000 rpm for 10 minutes, and the supernatant used to run the tests. The tests were run according to the manufacturer's suggestions. The plates were read on a Plate Reader Elx808 (Biotek, Vermont) at 450 nm wavelength. Total protein is measured using a bicinchoninic acid (BCA) protein assay kit assessing colorimetric detection and quantitation of total protein, read at 590 nm (Pierce, Rockford, IL). Protein levels within each group were reported as mean ± SE pg/mL. Plasma plasminogen activator inhibitor-1 (PAI-1) Determination At the time of euthanasia, blood samples were collected by intracardiac puncture. Blood was drawn into a syringe containing 3.8% sodium citrate (9:1 v:v), placed in an eppendorf tube, and immediately centrifuged at 3,000 ×g for 15 minutes. Plasma was snap frozen in liquid nitrogen then stored at minus 70 degrees centigrade. On the morning of the determination, plasma was thawed and active plasma PAI-1 determined using a commercially available kit (Molecular Innovations RPAIKT, Southfield, MI) that utilized an Enzyme-Linked ImmunoSorbent Assay (ELISA). These data were evaluated (ng/mL) and the means ± SEM and p-values were subsequently calculated. Murine Platelet and Leukocyte Microparticle Analysis Whole blood (400 μL) was removed from mice by intracardiac puncture using a syringe primed with 10% acid citrate dextrose. Blood was placed in an eppendorf tube centrifuged at 1,500 ×g and 23 °C for 25 minutes, plasma transferred to an additional eppendorf tube and spun again for 2 minutes at 15,000 ×g to obtain platelet poor plasma (PPP). Two hundred micro liters (200 μL) of PPP obtained from each mouse was diluted 1:3 with 600 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Additionally, 100 μL of individual mouse plasma was pooled and divided into three equal volumes and added to 600 μL of HEPES buffer for internal controls. All samples were centrifuged for 2 hours at 200,000 ×g to separate the microparticles (MPs). The supernatant was removed, and pelleted MPs were resuspended in HEPES buffer. Rat anti-murine PE (Chemicon International, Temecula, CA), or control rat PE IgG (BD Pharmingen, San Diego, CA), and rat anti-murine fluorescein isothiocyanate (BD Pharmingen) or control rat FITC IgG (BD Pharmingen) were added to samples to stain leukocytes and platelets. Murine antibody controls consisted of the following: FITC/PE, 1.5 μL/ 0.6 μL (negative control), leukocyte marker MAC-1 (PE), 12.0 μL (positive control), and platelet marker FITC (CD41), 1.2 μL (positive control). For MP quantification, a known quantity (250,000) of 3.0 – 3.4 µm beads (SPHERO™, BD Pharmingen) was added to each fixed sample prior to FACS analysis, and acquisition was stopped after 50,000 beads were counted in the R-1 bead gate. The diameter of these beads allowed discrimination from the MP population (1.0 µm or less) on light scatter and were counted using a separate bead gate as previously described [12]. Circulating Tissue Factor Activity Two hundred microliters of platelet poor plasma (PPP) was obtained by centrifuging blood at 1,500 ×g for 25 minutes. The plasma was removed and centrifuged once more for 2 minutes at 15,000 ×g to remove contaminating cells from the plasma. The tissue factor assay
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Statistical Evaluation and Animal Use All analyses were performed using SigmaStat (v.2.03). The statistical differences between groups were determined by the Student's t-test. Non-parametric data was assessed using the MannWhitney Rank Sum. A value of P ≤ .05 was considered significant and data reported as mean ± SE. All mice were housed and cared for by the University of Michigan Unit for Laboratory Animal Medicine and were free of pathogens. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals [19]. The University of Michigan Committee on Use and Care of Animals approved this research protocol. Results Fig. 1. Thrombus mass was significantly increased in OLD mice compared to YOUNG animals 2 days post venous thrombosis. YOUNG non-thrombosed control animals had significantly larger venous thrombi than OLD mice.
kit (American Diagnostica, Stamford, CT) combined soluble factor VIIa, Human Factor X, TF/TFPI free plasma, and the MP sample (spun down as above, but the pellet was resuspended in TF/TFPI free plasma). This combination activated Factor X, which then hydrolyzed Spectrozyme Xa (American Diagnostica Inc., Greenwich, CT). This hydrolysis occurred in a linear fashion at 37 °C as read on a spectrophotometer. Enzyme activity was determined by measuring the increase in absorbance of the free chromophore (pNA) generated per unit time at λ405 nm. The positive control was a known amount of human tissue factor (standard curve) while the negative control was microparticle free buffer. Human reagents were used to evaluate mouse tissue factor activity for comparative data analysis only.
Aging Increases Thrombus Formation On day 2, post-thrombosis, all mice were euthanized and their vein wall and associated thrombus were weighed and normalized to the length of the affected vein wall segment to evaluate thrombus mass. OLD mice (n = 8) demonstrated significantly larger venous thrombi than YOUNG animals (n = 8) (389 ± 18 vs. 336 ± 14 g × 10− 4/cm, P b.05) [Fig. 1]. Older Mice Have Decreased Vein Wall Inflammatory Cells OLD mice (n= 8) had significantly fewer vein wall monocyte (4± 0.4 vs.12 ± 2 cells/HPFs, P b.01), lymphocyte (1± 0.1 vs. 5 ± 2 cells/HPFs, P b.05), and total inflammatory cell populations (16 ± 3 vs. 29 ± 5 cells/ HPFs, P b.05) than YOUNG mice (n= 8), 2 days post-thrombosis (Fig. 2). No significant differences existed between OLD and YOUNG nonthrombosed control animals (data not shown).
Microparticle Re-injection Experiments
Vein Wall Protein Determination
Donor MP Preparation Mice from both OLD (n = 23) and YOUNG (n = 21) experimental groups underwent IVC ligation to induce thrombosis, as previously described [16,17]. Two days post-thrombosis, mice had whole blood withdrawn for the plasma microparticle assay, as previously described [12]. Mice were then euthanized and their IVC and associated thrombus weighed. The platelet poor plasma (PPP) was pooled from donor animals within each respective group and thoroughly mixed into two separate heterogeneous stock solutions. These stock solutions were re-aliquoted in equal amounts, snap frozen in liquid nitrogen − 196 °C, and then stored at −70 °C. A single aliquot of donor PPP was analyzed by flow cytometry to determine the total MP concentration of 160,000 MPs/200 µL of PPP. Prior to injection into recipient mice, MP aliquots were thawed and centrifuged in a 1:3 ratio with HEPES buffer pH 7.4 at 4 °C for 2 hours at 200,000 ×g. The supernatant was removed and pelleted MPs were resuspended in 100 µL of a 10 mM HEPES, 136 mM NaCl, 5 mM MgCl2, 50 mM KCl (pH 7.4) buffer for injection into recipient animals.
Aging Increases Vein Wall P-selectin OLD non-thrombosed control mice showed a trend of elevated vein wall P-selectin versus YOUNG mice. Two days post thrombosis, OLD thrombosed mice (n= 8) showed a statistically significant increase in vein wall P-selectin levels compared to YOUNG animals (n= 8) [7306 ± 938 vs. 3805 ± 745 pg/mL, P b.05] (Fig. 3).
Recipient Animal Preparation Recipient mice from OLD (n = 20) and YOUNG (n = 12) groups were anesthetized with isoflurane gas. 100 µL of the heterogeneous MP solution (160,000 MPs/100 µL of PPP) was administered into the penile vein. This concentration of MPs re-injected into recipient mice consistently produces venous thrombosis comparable to control mice that have undergone complete IVC ligation [18]. After the MP injection, the mice were surgically prepped and underwent IVC ligation. Two days post IVC thrombosis, the mice were euthanized and their IVC and associated thrombus were removed, weighed and normalized to length (thrombus mass) as previously described [16].
Plasma Plasminogen Activator Inhibitor-1 (PAI-1) Concentration Increases with Age Active plasma PAI-1 was measured in samples from both nonthrombosed controls and animals 2 days post thrombosis. In mice with thrombus present for 2 days, active plasma PAI-1 concentrations
Fig. 2. OLD mice had significantly fewer monocytes, lymphocytes, and total inflammatory cell populations migrating through the vein wall versus YOUNG animals.
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0.8 grams × 10− 4/cm/animal weight, P b.01) [Fig. 6]. Therefore, young recipient animals receiving microparticles from either young or old donors showed significant increases in thrombus mass when indexed to animal weight two days post thrombosis.
Discussion
Fig. 3. OLD thrombosed mice showed a significant increase in vein wall P-selectin levels compared to YOUNG animals post-thrombosis.
were significantly increased in OLD mice (n = 6) versus YOUNG animals (n = 6) [20 ± 4 vs. 8 ± 2 ng/mL, P b.05] (Fig. 4). Circulating Leukocyte-derived Microparticles Increase with Age OLD thrombosed animals (n = 10) displayed significantly higher circulating leukocyte-derived microparticles (MAC-1) than YOUNG mice (n = 9) [5817 ± 850 vs. 2564 ± 283 MPs/200 µL PPP, P b.01]. Platelet-derived (CD41) and total microparticles were non-significantly increased in OLD mice compared to YOUNG animals (Fig. 5A). There were no baseline differences between OLD and YOUNG nonthrombosed control groups evaluated for plasma microparticles. Microparticle Tissue Factor Activity Increases in Older Mice Mice from both OLD and YOUNG experimental groups had blood withdrawn for plasma MP assays 2 days post venous thrombosis. Plasma MPs from each mouse were then assayed in duplicate for TF procoagulant activity. OLD mice (n = 17) had plasma MPs with significantly higher TF activity 2 days post-thrombosis when compared to YOUNG (n = 11) animals (34 ± 4 vs. 24 ± 2 pM, P b.05) [Fig. 5B]. Younger Mice Receiving Microparticles Have Increased Thrombotic Potential In order to determine the acute thrombotic potential of MPs, murine MPs from OLD and YOUNG donor pools were re-injected into age-matched recipient mice at the time of thrombus induction with thrombus size evaluated 2 days later. Recipient animals received a concentration of 160,000 MPs in 100 µL of PPP from the age-matched donor pool and then underwent IVC thrombosis. Two days post reinjection of donor MPs, mice were euthanized for sample analysis. Both donor and recipient animals had their thrombus mass divided by animal body weight (grams) to normalize for size differences between young and older mice. When MPs from OLD donor animals, 2 days post thrombosis, were re-injected into YOUNG recipient animals (OLD-D into YOUNG-R, n = 5), there was an increased thrombus burden versus OLD recipient animals receiving MPs from OLD donors (OLD-D into OLD-R, n = 11) or YOUNG donor mice (YOUNG-D into OLD-R, n = 9), 2 days post thrombosis, (17 ± 1 vs. 12 ± .0.4 grams ± 10− 4/cm/animal weight, P b.01,13 ± 0.8 grams × 10− 4/cm/animal weight, P b.05). Microparticles from YOUNG donor mice re-injected into YOUNG recipients (YOUNG-D into YOUNG-R, n = 7) had significantly larger venous thrombi when compared to older mice receiving MPs from OLD (OLD-D into OLD-R, n = 11) or YOUNG (YOUNG-D into OLD-R, n = 9) donor mice, 2 days post thrombosis (16 ± 0.7 vs.12 ± .0.4, 13 ±
Using a mouse model of VT, our data showed that aging was associated with increased thrombus size in mice. We reported that several prothrombotic circulating and vein wall factors were elevated in older animals when compared to young mice undergoing stasis induced thrombosis. These included: 1) decreased vein wall inflammatory cells; 2) increases plasma PAI-1; 3) increased vein wall P-selectin; 4) increased leukocyte MPs levels; 5) increased MP tissue factor activity. Importantly, the donor/recipient experiments suggest the host age and physiological environment are important factors in stasis thrombus resolution. Older mice had significantly greater thrombus mass when compared to younger animals. Consistently, OLD animals had significantly decreased vein wall inflammatory cell populations, which may affect thrombus resolution in these animals [20]. In a previous natural history study using the same IVC ligation model of venous thrombosis, we documented an early neutrophil (PMN) cell vein wall inflammatory cell influx followed by a later influx of monocytes during thrombosis [16]. Neutrophils and monocytes both have a defined role in inflammation and thrombus resolution. For example, in a rat IVC ligation model of venous thrombosis, neutrophil depletion of these animals promoted larger venous thrombi associated with increased thrombus fibrosis in animals evaluated at day 2 and day 7 [21]. PMNs are necessary for early thrombus resolution by promoting the lysis of both fibrin and collagen [22,23]. Inflammatory cells, especially monocytes, have been shown to promote thrombolysis due to their secretion of proteolytic enzymes [24–26]. Post thrombosis, increasing levels of monocyte chemotactic protein-1 (MCP-1) stimulate chemotaxis and activation of monocytes which promote venous thrombus resolution [27] . In this study, YOUNG animals with significantly greater vein wall inflammatory cell populations had significantly smaller venous thrombi. PAI-1 inhibits the serine proteases tPA and uPA, and thus fibrinolysis, the process which promotes thrombus resolution. OLD thrombosed animals had a significant increase in circulating PAI-1 activity compared to YOUNG. PAI-1 has been shown to significantly increase with age in both plasma and murine tissues and in aged rats, which supports our findings [28–30]. Recent clinical studies have reviewed the relationship between aging and increased circulating levels of PAI-1. With age, factors such as obesity and stress, promote increased production of PAI-1 levels [6,28,29,31,32]. In this study,
Fig. 4. Active plasma plasminogen activator inhibitor-1 (PAI-1) in OLD mice was significantly elevated at 2 days post thrombosis when directly compared to YOUNG thrombosed animals at the same time point.
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Fig. 5. A. OLD thrombosed animals displayed significantly higher circulating leukocyte-derived microparticles (MAC-1) than YOUNG mice. B: OLD mice had plasma microparticles with significantly higher tissue factor activity 2 days post-thrombosis versus YOUNG mice.
significantly increased PAI-1 activity in OLD mice suggests reduced fibrinolysis, which may explain the causal relationship with the larger thrombi observed in the OLD mice. Previous studies by our laboratory and others have shown that increases in P-selectin and interactions of P-selectin with its receptor, P-selectin glycoprotein-1 (PSGL-1), promote leukocyte rolling and
Fig. 6. To determine the thrombotic potential of microparticles (MPs), murine MPs from OLD and YOUNG donor pools of animals were re-injected into recipient mice of different ages 2 days post-thrombosis. Two days post re-injection of MPs into recipient mice; animals were humanely euthanized and had their thrombus mass evaluated. A significant increase in thrombus burden in YOUNG recipient animals was noted.
emigration at the site of injured vascular endothelium encouraging thrombogenesis [7,10,33]. Using rodent models of venous thrombosis to evaluate P-selectin inhibition, we have documented that thrombosis can be inhibited without total inhibition of vein wall inflammatory cells [17,34]. These data suggest that other inflammatory mechanisms, in addition to P-selectin promote inflammatory cell extravasation through the vein wall. Thus, the amount of P-selectin and vein wall inflammatory cells does not correlate. This lack of correlation was evident in a study testing the effects of a small molecule inhibitor of Pselectin on venous thrombosis. In this rodent study, P-selectin inhibition effectively decreased vein wall fibrosis without decreasing vein wall inflammatory cells extravasation [34]. Another contributor to the increased procoagulant state of the OLD mice may be the significantly increased levels of circulating plasma leukocyte-derived MPs. With damage to the venous endothelium, Pselectin becomes expressed on endothelial cells and platelets simulating interactions between P-selectin and its receptor, PSGL-1. P-selectin: PSGL-1 interactions produce the generation of MPs from leukocytes post vascular injury [7,8,10,12]. Such interactions activate procoagulant MP release from platelets, leukocytes and endothelial cells. Tissue factor is primarily responsible for thrombosis. Work by Nemerson determined that TF is naturally circulating on a subpopulation of microparticles [35]. These TF-bearing MPs have been shown to accumulate in the developing thrombus after vascular injury [15,28,36]. This mechanism is driven by P-selectin: PSGL-1 interactions [9,10,36]. In the current study, mice had plasma MPs evaluated for circulating TF activity 2 days post venous thrombogenesis. OLD animals had significantly higher concentrations of MP TF activity than YOUNG animals. These findings suggest that leukocyte MPs have increased TF activity, and subsequently increases thrombosis.
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Although our laboratory and others have consistently been able to detect mouse plasma tissue factor activity using human reagents, we recognize that mouse TF may not have the same specificity or interactions as human TF. It has been shown that human TF interacts effectively with both human and mouse VIIa [37,38]. However the converse is not true, mouse TF interacts poorly with human VIIa [37,38]. Therefore, it is possible that the true mouse plasma TF activity in this study was underreported in our OLD and YOUNG mice 2 days post thrombosis. Presently, multiple leaders in the field of tissue factor biology are working to develop specific murine tissue factor antibodies in an effort to standardize murine in vitro and in vivo TF assays. Future studies will also need to evaluate the role of latent/ encrypted TF during acute venous thrombosis. To determine if circulating MPs or the age of the recipient determines the thrombogenic response, we re-injected MPs obtained from donor animals 2 days post-thrombosis into age-matched recipient mice. Previous work in our laboratory has determined that acute inflammatory responses (increased vein wall inflammatory cell extravasation and P-selectin protein levels) are peaking at 2 days post venous thrombosis in our mouse model [16,17]. Thus, the 2 day post thrombosis time-point has documented pro-inflammatory activity present. YOUNG recipient animals had significantly larger venous thrombi 2 days post-injection of MPs regardless if the re-injected MPS were from YOUNG or OLD donor animals. This finding did not hold true for OLD recipient mice receiving donor MPs from OLD or YOUNG mice. In previous studies, mice with TFpositive MPs from mice with high circulating levels of P-selectin, showed that these MPs were incorporated into developing thrombi [11]. From these data, it can be hypothesized that MPs are playing a role in the development of large thrombi in YOUNG animals. This was an unexpected finding in this study. Based on the other factors identified in this study, we hypothesized the MPs re-injected into older animals with impaired mechanisms of thrombolysis (significantly elevated circulating active PAI-1), should have larger venous thrombi. It is clear that other mechanisms are at play such as host environment (blood and endothelium) and duration of thrombosis. Mice were evaluated 2 days post thrombosis which is an early time point for this dynamic process. Future experiments warrant evaluating both early and late thrombotic events in animals at different ages to determine the true mechanism behind these results. The phenotype of YOUNG animals appears to promote the development of thrombi of the same magnitude in these re-injection experiments. This suggests that the recipient's physiological environment is more important than the age of the donor of MPs. We did not formally evaluate coagulation tests in this study however, there were no adverse effects such as wound healing or bleeding complications detected in either OLD or YOUNG mice. In conclusion, we have shown differences in several circulating and vein wall factors that influence thrombosis between OLD and YOUNG animals in our rodent model. We hypothesize that with increasing age changes occur in both the venous endothelium and circulation that promote a prothrombotic environment. These factors include increased activity in PAI-1 and procoagulant MPs, a sub-population that has measured TF activity. In addition, there are active mechanisms that acutely modulate vein wall inflammatory cell populations while favoring P-selectin expression producing a thrombogenic vascular environment in OLD animals. These data collectively suggest possible mechanisms influencing the increase in VT that occurs with aging. Conflicts of interest The authors of this manuscript have no conflicts of interest to disclose. Acknowledgements Financial support was obtained from the Diversity & Career Development Office (DCDO) [to DDM], Esperance Family Foundation
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(to APM), and Department of Surgery University of Michigan (to DDM). We thank Ms. Iris Richardson for her technical assistance. References [1] Cushman M, Tsai AW, White RH, Heckbert SR, Rosamond WD, Enright P, et al. Deep vein thrombosis and pulmonary embolism in two cohorts: the longitudinal investigation of thromboembolism etiology. Am J Med 2004;117:19–25. [2] Heit JA. Venous thromboembolism: disease burden, outcomes and risk factors. J Thromb Haemost 2005;3:1611–7. [3] Silverstein MD, Heit JA, Mohr DN, Petterson TM, O'Fallon WM, Melton III LJ. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Arch Intern Med 1998;158:585–93. [4] Tsai AW, Cushman M, Rosamond WD, Heckbert SR, Polak JF, Folsom AR. Cardiovascular risk factors and venous thromboembolism incidence: the longitudinal investigation of thromboembolism etiology. Arch Intern Med 2002;162:1182–9. [5] Goldhaber SZ. Pulmonary embolism. Lancet 2004;363:1295–305. [6] Yamamoto K, Takeshita K, Kojima T, Takamatsu J, Saito H. Aging and plasminogen activator inhibitor-1 (PAI-1) regulation: implication in the pathogenesis of thrombotic disorders in the elderly. Cardiovasc Res 2005;66:276–85. [7] Andre P, Hartwell D, Hrachovinova I, Saffaripour S, Wagner DD. Pro-coagulant state resulting from high levels of soluble P-selectin in blood. Proc Natl Acad Sci U S A 2000;97:13835–40. [8] Cambien B, Wagner DD. A new role in hemostasis for the adhesion receptor P-selectin. Trends Mol Med 2004;10:179–86. [9] Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, et al. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle Pselectin glycoprotein ligand 1 and platelet P-selectin. J Exp Med 2003;197:1585–98. [10] Furie B, Furie BC. Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation. Trends Mol Med 2004;10:171–8. [11] Hrachovinova I, Cambien B, Hafezi-Moghadam A, Kappelmayer J, Camphausen RT, Widom A, et al. Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A. Nat Med 2003;9:1020–5. [12] Myers DD, Hawley AE, Farris DM, Wrobleski SK, Thanaporn P, Schaub RG, et al. Pselectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg 2003;38:1075–89. [13] Myers Jr DD, Wrobleski SK, Longo C, Bedard PW, Kaila N, Shaw GD, et al. Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation. Thromb Haemost 2007;97:400–7. [14] Rectenwald JE, Myers Jr DD, Hawley AE, Longo C, Henke PK, Guire KE, et al. Ddimer, P-selectin, and microparticles: novel markers to predict deep venous thrombosis. A pilot study. Thromb Haemost 2005;94:1312–7. [15] Giesen PL, Rauch U, Bohrmann B, Kling D, Roque M, Fallon JT, et al. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci U S A 1999;96:2311–5. [16] Myers Jr D, Farris D, Hawley A, Wrobleski S, Chapman A, Stoolman L, et al. Selectins influence thrombosis in a mouse model of experimental deep venous thrombosis. J Surg Res 2002;108:212–21. [17] Myers Jr DD, Rectenwald JE, Bedard PW, Kaila N, Shaw GD, Schaub RG, et al. Decreased venous thrombosis with an oral inhibitor of P selectin. J Vasc Surg 2005;42:329–36. [18] Ramacciotti E, Hawley AE, Farris DM, Ballard NE, Wrobleski SK, Myers Jr DD, et al. Leukocyte- and platelet-derived microparticles correlate with thrombus weight and tissue factor activity in an experimental mouse model of venous thrombosis. Thromb Haemost 2009;101:748–54. [19] Institute of Laboratory Animal Resources CoLS, National Research Council. Guide for the care and use of laboratory animals. 7th ed. Washington, D.C.: National Academy Press; 1996. [20] Henke PK, Varma MR, Deatrick KB, Dewyer NA, Lynch EM, Moore AJ, et al. Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization. Thromb Haemost 2006;95:272–81. [21] Varma MR, Moaveni DM, Dewyer NA, Varga AJ, Deatrick KB, Kunkel SL, et al. Deep vein thrombosis resolution is not accelerated with increased neovascularization. J Vasc Surg 2004;40:536–42. [22] Stewart GJ. Neutrophils and deep venous thrombosis. Haemostasis 1993;23 (Suppl 1):127–40. [23] Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, et al. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg 2003;38:1090–8. [24] Burnand KG, Gaffney PJ, McGuinness CL, Humphries J, Quarmby JW, Smith A. The role of the monocyte in the generation and dissolution of arterial and venous thrombi. Cardiovasc Surg 1998;6:119–25. [25] Henke PK, Varga A, De S, Deatrick CB, Eliason J, Arenberg DA, et al. Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol 2004;24:1130–7. [26] Humphries J, McGuinness CL, Smith A, Waltham M, Poston R, Burnand KG. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg 1999;30:894–9. [27] Hogaboam CM, Steinhauser ML, Chensue SW, Kunkel SL. Novel roles for chemokines and fibroblasts in interstitial fibrosis. Kidney Int 1998;54:2152–9. [28] Chou J, Mackman N, Merrill-Skoloff G, Pedersen B, Furie BC, Furie B. Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation. Blood 2004;104:3190–7. [29] Hashimoto Y, Kobayashi A, Yamazaki N, Sugawara Y, Takada Y, Takada A. Relationship between age and plasma t-PA, PA-inhibitor, and PA activity. Thromb Res 1987;46:625–33. [30] Takeshita K, Yamamoto K, Ito M, Kondo T, Matsushita T, Hirai M, et al. Increased expression of plasminogen activator inhibitor-1 with fibrin deposition in a murine model of aging, "Klotho" mouse. Semin Thromb Hemost 2002;28:545–54.
78
A.P. McDonald et al. / Thrombosis Research 125 (2010) 72–78
[31] Mari D, Coppola R, Provenzano R. Hemostasis factors and aging. Exp Gerontol 2008;43:66–73. [32] Yamamoto K, Takeshita K, Shimokawa T, Yi H, Isobe K, Loskutoff DJ, et al. Plasminogen activator inhibitor-1 is a major stress-regulated gene: implications for stress-induced thrombosis in aged individuals. Proc Natl Acad Sci U S A 2002;99:890–5. [33] Myers DD, Wakefield TW. Inflammation-dependent thrombosis. Front Biosci 2005;10:2750–7. [34] Myers Jr DD, Henke PK, Bedard PW, Wrobleski SK, Kaila N, Shaw G, et al. Treatment with an oral small molecule inhibitor of P selectin (PSI-697) decreases vein wall injury in a rat stenosis model of venous thrombosis. J Vasc Surg 2006;44:625–32.
[35] Nemerson Y. Tissue factor and hemostasis. Blood 1988;71:1–8. [36] Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 2002;8:1175–81. [37] Fang CH, Lin TC, Guha A, Nemerson Y, Konigsberg WH. Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3. Thromb Haemost 1996;76:361–8. [38] Janson TL, Stormorken H, Prydz H. Species specificity of tissue thromboplastin. Haemostasis 1984;14:440–4.