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Thomas W Wakefield

Publications and source records attributed to Thomas W Wakefield.

At least 37 records · Page 2Linked to original sources
PubMed2005

Vein wall remodeling after deep vein thrombosis involves matrix metalloproteinases and late fibrosis in a mouse model.

HYPOTHESIS: Deep venous thrombosis (DVT) confers vein wall injury associated with fibrosis and extracellular matrix (ECM) turnover, likely mediated by matrix proteases. This study investigated the expression of proteases and collagen involved in early vein wall remodeling. METHODS: In the mouse, DVT was produced by ligation of the infrarenal inferior vena cava (IVC) or sham operation, and tissue was harvested at 4, 8, and 12 days. The vein wall tissue was processed for real-time reverse transcriptase-polymerase chain reaction (6 to 8 per time point), Western immunoblotting (5 per time point), and gelatin zymography (5 per time point). Analysis of variance was used for multiple comparisons, and a P < .05 was significant. RESULTS: Thrombus resolution was documented by a 38% decrease in the thrombosed IVC weight from day 4 to day 12 (P = .007). Total vein wall collagen increased over time, with a corresponding increase in procollagen I and III, and expression peaked at 12 days (24-fold and 6.1-fold, respectively, P < .02). Matrix metalloproteinase-2 (MMP-2) gene expression was 23-fold greater at 12 days after thrombus formation compared with sham or 4 days after thrombosis (P < .05). Total MMP-2 activity was also significantly elevated at 12 days compared with sham (P < .05). MMP-9 expression was 19-fold and 27-fold higher at days 4 and 8, respectively, relative to sham (P < .05), with no difference in activity. MMP-14 expression was twofold to 3.6-fold greater at day 12 compared with earlier time points and shams (P < .001), but no differences in protein levels were found. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein levels were not significantly different from sham over time; however, the ratio of uPA to PAI-1 was decreased through 8 days. CONCLUSIONS: Vein wall remodeling after DVT is similar to wound healing and is associated with increased procollagen gene expression and total collagen. It is also associated with increased early MMP-9 expression, followed by MMP-2 expression and activity after DVT resolution. CLINICAL RELEVANCE: Deep vein thrombosis is an often neglected problem that long term is associated with the postphlebitic syndrome of limb swelling, pain, and often ulceration. The basic mechanisms of the vein wall damage that results have not been delineated. The following study describes the vein wall matrix metalloproteinase gene and activity response induced over time in the vein wall after DVT. Additionally, the corresponding collagen upregulation and proximate plasmin system mediators are determined. With this knowledge, potential therapies to reduce vein wall injury directly might be possible.

Animals↗
PubMed2005

Decreased venous thrombosis with an oral inhibitor of P selectin.

BACKGROUND: P-selectin inhibition with protein therapeutics such as antibodies or soluble ligands given intravenously can decrease thrombosis in a mouse ligation model of venous thrombosis. In this study, we hypothesized that oral inhibition of P selectin with a novel oral nonprotein inhibitor (PSI-697) would decrease thrombosis and circulating microparticle populations. This study evaluated the effects on thrombosis and circulating microparticle populations in this murine venous thrombosis model. METHODS: Mice underwent inferior vena cava ligation to induce thrombosis. Mice with high circulating level of P selectin, Delta Cytoplasmic Tail (CT), mice gene-deleted for both E- and P-selectin knockout (EPKO), and wild-type C57BL/6 mice (WT) were studied without and with administration of PSI-697 in food (100 mg/kg daily) from 2 days before thrombosis until the end of the study. Animals were killed 2 and 6 days later. Evaluations included thrombus weight (TW), vein wall morphometrics, microparticle quantification by using fluorescence-activated cell sorter analysis, and vein wall enzyme-linked immunosorbent assays for interleukin (IL)-10, P selectin, and monocyte chemotactic protein 1. RESULTS: PSI-697 significantly decreased TW in WT and CT mice, with a treated vs nontreated TW of 132 +/- 24 vs 228 +/- 29 x 10(-4) g (P = .014) and 166 +/- 19 vs 281 +/- 16 x 10(-4) g (P = .001), respectively. At day 6, the effect was significant only in the CT group (P < .05). Drug therapy at day 2 significantly increased vein wall monocytes in WT mice and increased monocytes and total inflammatory cells in CT animals. A significant decrease in neutrophils and total inflammatory cells was seen in EPKO mice at day 2 with therapy. Therapy significantly increased platelet-derived microparticles and total microparticles in CT mice on day 2. Changes in treated WT and treated EPKO animals were not significant compared with respective vehicle treatments at day 2. On day 6, therapy significantly decreased total microparticles in EPKO animals. Vein wall expression of IL-10 increased in all groups with therapy at day 2 (n = 18) and was significantly increased in WT (2687.5 +/- 903 pg/mL vs 636 +/- 108 pg/mL total protein; P = .038) and CT (2078 +/- 295 pg/mL vs 432 +/- 62 pg/mL total protein; P = .001) mice. Therapy significantly decreased vein wall P selectin, monocyte chemotactic protein 1, and IL-10 levels at day 6. CONCLUSIONS: PSI-697 decreased thrombosis. P-selectin inhibition allowed vein wall inflammatory cell extravasation in this model of complete ligation. Circulating microparticles (platelet-derived microparticles and total microparticles) increased with P-selectin inhibition, possibly because of decreased consumption into the thrombus. In summary, the oral administration of an inhibitor to P selectin provides significant TW reduction. CLINICAL RELEVANCE: Deep venous thrombosis is a significant national health problem in the general population. The average annual incidence of deep venous thrombosis is approximately 250,000 cases per year. The selectin family of adhesion molecules is thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. Recent studies have explored the possible therapeutic implications of P-selectin inhibition to modulate venous thrombosis. For example, prophylactic dosing of a recombinant P-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. Additionally, treatment of 2-day iliac thrombi with a recombinant protein, P-selectin inhibitor, significantly improves vein reopening in nonhuman primates. It is interesting to note that P-selectin inhibition decreases thrombosis without adverse anticoagulation. On the basis of the results from these previous studies, the use of P-selectin antagonism is a logical therapeutic approach to treat venous thrombosis. All inhibitors developed to date are either proteins or small molecules with low oral bioavailability that require intravenous or subcutaneous injection. This study evaluates, for the first time, a novel orally bioavailable inhibitor of P-selectin (PSI-697).

Animals↗
PubMed2005

Correspondence of ultrasound elasticity imaging to direct mechanical measurement in aging DVT in rats.

Previous ultrasound elasticity imaging experiments supported a generally accepted concept that the hardness of deep venous thrombi increases with thrombus aging. Results also showed that this noninvasive imaging technique can accurately predict thrombus age through strain estimates, in a well-controlled animal study. In the present study, as an alternative means to characterize elastic properties of thrombi, we used a direct mechanical measurement system to estimate Young's modulus of ex vivo thrombi. Unlike conventional indentation tests, the device uses a specific compression geometry for cylindrical tissue specimens. We also proposed an approximation scheme to retrieve Young's modulus from force-displacement measurements made using the device. Finite element simulations and calibrations on tissue-mimicking phantoms validated the system. Then, using two groups of rats with surgically-induced thrombi, we further investigated the correlation between Young's modulus measured ex vivo and elasticity images reconstructed in vivo. This comparison was accomplished by converting the intrathrombus strains measured in the in vivo studies into Young's modulus estimates using a model-based approach. Good agreement between time-dependent Young's modulus estimates observed in vivo and direct measurements of Young's modulus using the mechanical device helps to confirm the ability of elasticity imaging to age deep venous thrombi for efficient treatment.

Aging↗
PubMed2005

The role of inflammation in early and late venous thrombosis: Are there clinical implications?

Venous thrombosis is associated with a significant inflammatory response. Inflammatory cells, adhesion molecules (especially selectins), cytokines, and procoagulant microparticles appear to be associated with the thrombogenic process. Once thrombus forms, inflammatory cells are important to thrombus resolution along with fibrinolytic agents and proinflammatory mediators. Collagen and elastin breakdown by the DVT renders the vein wall stiff and non-compliant. Rapid and complete thrombus resolution should lessen vein wall damage and lessen or prevent the development of chronic venous insufficiency. Understanding the basic biology of thrombogenesis and thrombus resolution is important, as novel therapies to both prevent and treat venous thrombosis and hasten thrombus resolution should result from a better understanding of the basic biological mechanisms.

Cell Adhesion Molecules↗
PubMed2005

Osteomyelitis of the foot and toe in adults is a surgical disease: conservative management worsens lower extremity salvage.

OBJECTIVE: To characterize the national epidemiology of adult osteomyelitis (OM) and, using a single institutions' experience, test the hypothesis that early surgical therapy as compared with antibiotics alone results in an improved chance of wound healing and limb salvage. BACKGROUND: Foot and digit OM is a very common problem for which management is variable and for which few guidelines exist. METHODS: The Nationwide Inpatient Sample (NIS) and a single institution review from 1993 to 2000 form the basis of this study, using ICD-9CM codes for lower extremity foot and digit OM. Demographics, risk factors, and treatments were analyzed against the outcomes of a healed wound, limb salvage, and death. RESULTS: The NIS included 51,875 patients (incidence = 9/10,000 patients per year) with a mean age of 60 years, and 59% were men. The median length of stay decreased from 9 to 6 days (P < 0.001), but the average admission charge of 19,000 dollars did not significantly decrease over 7 years. Of these patients, 23% underwent a digit amputation and 8.5% suffered proximal limb loss. Single-institution analysis of 237 consecutive patients with OM confirmed a similar mean age (58 years), gender (67% men), and most presented with a foot or digit ulcer (56%). Wound healing was achieved in 56% and overall limb salvage was 80%. Decreased wound healing was associated with peripheral vascular occlusive disease (odds ratio, 0.4; 95% confidence interval, 0.2-0.8, P = 0.006) and preadmission antibiotic use (odds ratio, 0.2; 95% confidence interval, 0.05-1.1, P=0.07), while surgical debridement (odds ratio, 2.2; 95% confidence interval, 1.2-4.2, P = 0.02) was associated with increased healing. Limb salvage was improved with an arterial bypass (odds ratio, 3.9; 95% confidence interval, 1.1-14, P = 0.04), while preadmission solid organ transplant (odds ratio, 0.37; 95% confidence interval, 0.14-0.96, P = 0.04), peripheral vascular occlusive disease (odds ratio, 0.25; 95% confidence interval, 0.12-0.5, P = 0.001), and preadmission antibiotic use (odds ratio, 0.34; 95% confidence interval, 0.15-0.77, P = 0.009) were associated with greater limb loss. CONCLUSION: Digit OM is an expensive and morbid disease. Aggressive surgical debridement/digit amputation and selected use of arterial bypass should improve wound healing and limb salvage, respectively. In contrast, antibiotic therapy alone is associated with decreased wound healing and limb salvage.

Aged↗
PubMed2005

D-dimer, P-selectin, and microparticles: novel markers to predict deep venous thrombosis. A pilot study.

Current plasma markers for diagnosis of deep venous thrombosis (DVT) allow for exclusion of the diagnosis, but lack adequate specificity to establish the diagnosis. Thus, a prospective study was performed to determine the sensitivity and specificity of plasma assays for D-dimer, soluble P-selectin (P-selectin), and total microparticles in patients with documented DVT by duplex ultrasound. Three groups of individuals were examined: 30 normals; 22 positive for DVT on duplex ultrasound (Group 2); and 21 symptomatic, but negative on duplex ultrasound for DVT (Group 3). Group 1 individuals had D-dimer values of 1.53 +/- 0.12 mg/l and P-selectin values of 0.34 +/- 0.05 ng/mg total protein. Group 2 vs. Group 3 individuals had D-dimer values of 7.57 +/- 2.03 vs. 3.19 +/- 0.79 mg/l, p = 0.02; P-selectin values of 0.98 +/- 0.11 vs. 0.55 +/- 0.08 ng/mg total protein, p < 0.01; and micro-particle values of 129 +/- 17% vs. 99 +/- 12% of control, p = ns. Using a logistic regression model with dichotomous variables, we determined a sensitivity of 73%, specificity of 81%, and accuracy of 77% when combining D-dimer, soluble P-selectin, and total microparticles to differentiate Group 2 from Group 3 patients. Logistic regression using continuous variables yielded similar results (p = 0.05). This study demonstrates that plasma markers for DVT can be developed and achieve moderate sensitivity and specificity in diagnosing DVT. However for clinical applicability, the sensitivity/specificity will need to be improved. These studies also suggest the importance of soluble P-selectin in assessing DVT in humans.

Female↗
PubMed2005

Gender differences in deep venous thrombosis in a rat model: a preliminary study.

The purpose of this study was to determine whether gender differences have an effect on inflammation and thrombosis in a rat model of venous thrombosis. A thrombus was created in mature female (n = 12) and male (n = 12) Sprague Dawley rats (Rattus norvegicus) by ligating the inferior vena cava (IVC). The IVC containing the thrombus was harvested at 1 and 3 days postligation, weighed, measured, and submitted for immunohistochemical analysis. In addition, hematology was performed at selected time points. There were no statistically significant differences in thrombus mass (mean +/- 1 standard deviation) between female and male rats at 1 (683 +/- 47.7 x 10(-4) versus 660 +/- 112.0 x 10(-4) g/cm) or 3 (683 +/- 83.3 x 10(-4) versus 580 +/- 86.0 x 10(-4) g/cm) days post-ligation. Females had significantly more platelets than did males on day 1 (741 +/- 37.2 versus 523 +/- 55.1 K/microL, P < 0.01). Day 3 males showed significant increases in vein wall neutrophils (18.0 +/- 2.30 versus 11.2 +/- 1.38, P < 0.05), ED-1-positive monocytes (54.4 +/- 16.0 versus 18.7 +/- 5.63, P < 0.05), and circulating white blood cells (15.4 +/- 0.947 x 10(3) versus 10.9 +/- 0.714 x 10(3)/microL, P < 0.01) at post-thrombosis when compared with females. We conclude that although female rats had greater thrombus mass, the male rats demonstrated more inflammatory cells in circulation and in their vein walls. This finding suggests that inflammation plays a role in thrombus resolution.

Animals↗
PubMed2004

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall.

Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF(-/-), hTF-Tg+, or "low-TF") demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

Animals↗
PubMed2004

Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model.

OBJECTIVE: To determine the role of CXCR2, the receptor for cysteine-X-cysteine (CXC) chemokines, and its primary effector cell, the neutrophil (PMN), on deep venous thrombosis (DVT) resolution. METHODS AND RESULTS: DVT in BALB/c, anti-CXCR2 antibody-treated, and BALB/c CXCR2(-/-) mice were created by infrarenal inferior vena cava (IVC) ligation and the thrombus harvested at various time points over 21 days. The CXCR2(-/-) mice had significantly larger thrombi at early time points (days 2 to 8), and significantly decreased intrathrombus PMNs, monocytes, and neovascularization as compared with controls. Thrombus KC/CXCL1 was significantly higher at 2 days in CXCR2-/- thrombi as measured by enzyme-linked immunosorbent assay. Fibrin content was significantly higher, with less uPA gene expression at 4 days in CXCR2-/- thrombi. Late fibrotic maturation of the thrombus was delayed in the CXCR2-/- mice, with significantly decreased 8 day MMP-2 activity, whereas MMP-9 activity was elevated as compared with controls. Similar impairment in DVT resolution was found at 8 days with anti-CXCR2 inhibition. However, systemic neutropenia, unlike CXCR2 deletion, did not increase the thrombus size as compared with controls. CONCLUSIONS: Normal DVT resolution involves CXCR2-mediated neovascularization, collagen turnover, and fibrinolysis, and it is probably primarily monocyte-dependent.

Animals↗
PubMed2004

Deep vein thrombosis resolution is not accelerated with increased neovascularization.

INTRODUCTION: Deep venous thrombosis (DVT) resolution involves fibrinolysis, neovascularization, and fibrosis. We hypothesized that promoting neovascularization would accelerate DVT resolution. METHODS: A rat model of stasis DVT was produced with proximal ligation of the inferior vena cava (IVC) and all visible tributaries. One microg of interferon inducible protein (IP-10; angiostatic chemokine), basic fibroblast growth factor (bFGF; pro-angiogenic cytokine), epithelial neutrophil activating protein (ENA-78; pro-angiogenic chemokine), or saline solution control was injected into the IVC after ligation, and then via tail vein injection daily until sacrifice at either 4 or 8 days. Peripheral blood counts were measured, and thrombus weight was recorded at sacrifice. Laser Doppler in vivo imaging was used to estimate post-thrombotic IVC blood flow. Immunohistologic assessment of the thrombosed IVC for polymorphonuclear neutrophils (PMNs), monocytes (ED-1), and laminin (neovascular channels) was performed or the thrombus was separated from the IVC and assayed for keratinocyte cytokine (KC), monocyte chemotactic protein-1 (MCP-1), bFGF with enzyme-linked immunosorbent assay (ELISA), and total collagen with a direct colorimetric assay. RESULTS: Peripheral blood and intrathrombus PMNs and monocytes were not significantly different in the treated or control rats. There were no differences in any measure at 4 days. At 8 days, thrombus neovascularity, but not weight or collagen content, was increased in rats treated with bFGF or ENA-78 compared with control rats (17.6 +/- 0.93, 16.2 +/- 0.97 vs 13.2 +/- 0.79; channels/5 high-power fields (hpf; n = 6-10; P <.05). Post DVT IVC blood flow was significantly increased in bFGF-treated rats but not in rats treated with IP-10 or ENA-78, as compared with control rats. Rats treated with ENA-78 had increased intrathrombus bFGF compared with control rats (85 +/- 27 pg/mg protein vs 20 +/- 6 pg/mg protein; n = 6; P <.05), but other mediators were not significantly different in treated rats compared with control rats. CONCLUSION: Pro-angiogenic compounds increase thrombus neovascularization, but this does not correlate with smaller or less fibrotic DVT. Mechanisms other than neovascularization may be more important to hasten DVT dissolution. Clinical relevance Improved therapy for deep venous thrombosis (DVT) will ideally increase the rate of thrombus dissolution and eliminate the bleeding risks of anticoagulation. This study evaluated promoting DVT neovascularization with angiogenic chemokines, and, while successful by experimental measures, this did not translate into smaller DVT. Solely promoting thrombus neovascularization will not likely speed resolution.

Animals↗
PubMed2004

Revision of the CEAP classification for chronic venous disorders: consensus statement.

The CEAP classification for chronic venous disorders (CVD) was developed in 1994 by an international ad hoc committee of the American Venous Forum, endorsed by the Society for Vascular Surgery, and incorporated into "Reporting Standards in Venous Disease" in 1995. Today most published clinical papers on CVD use all or portions of CEAP. Rather than have it stand as a static classification system, an ad hoc committee of the American Venous Forum, working with an international liaison committee, has recommended a number of practical changes, detailed in this consensus report. These include refinement of several definitions used in describing CVD; refinement of the C classes of CEAP; addition of the descriptor n (no venous abnormality identified); elaboration of the date of classification and level of investigation; and as a simpler alternative to the full (advanced) CEAP classification, introduction of a basic CEAP version. It is important to stress that CEAP is a descriptive classification, whereas venous severity scoring and quality of life scores are instruments for longitudinal research to assess outcomes.

Chronic Disease↗
PubMed2004

Staging deep venous thrombosis using ultrasound elasticity imaging: animal model.

Deep venous thrombi undergo progressive hardening with age. However, the evolution rate remains poorly characterized by both invasive and noninvasive techniques. In a previous study (Emelianov et al. 2002), we demonstrated the potential of ultrasound elasticity imaging to noninvasively detect and age thrombus using a rat-based model. Knowing that thrombi harden over time is useful, but the value of the technique relies on whether the age of a thrombus can be predicted from strain estimates, and how accurate these predictions are. The objective of the present study is to answer these two questions. In the previous study, thrombus elasticity changes were monitored only on day 3, 6 and 9 after surgically induced formation of thrombosis in rat inferior vena cavas. In this study, ultrasound elasticity imaging was performed on two independent groups of rats (16 in total) starting from day 3 through day 10 with more temporal samples through the thrombus maturation process. For each rat, thrombus hardness was quantified at each scan interval by measures of normalized strains and reconstructed relative Young's moduli. In both groups, strain magnitudes exhibit progressive decrease as clots age. The relationship between the normalized strain and the clot age was developed from the first group and evaluated by the second group. Statistical analysis showed that the age estimation accuracy is within 0.8 day. If further research can successfully transfer the animal clot-hardening model to human patients, we believe that elasticity imaging will become a key component of venous compression ultrasound for effective diagnosis and treatment of deep venous thrombosis.

Animals↗
PubMed2003

Cellular IL-10 is more effective than viral IL-10 in decreasing venous thrombosis.

BACKGROUND: Systemic administration of cellular interleukin-10 (cIL-10) and gene transfection of viral interleukin-10 (vIL-10) at thrombus induction decreases vein wall inflammation. Only cIL-10, despite sharing an 84% amino acid sequence homology with vIL-10, decreases thrombosis through mechanisms yet to be determined. METHODS: C57BL/6 mice (Mus musculus, n99) were studied. Inferior vena caval thrombosis was created by inferior vena caval ligation and the animals were sacrificed and evaluated at days 2 and 6 after ligation. At thrombus induction groups received intravenous 0.25 microg of cIL-10, 0.25 microg of vIL-10, or saline (untreated controls). Evaluations included thrombus mass and vein wall leukocyte counts, protein levels, and reverse-transcription polymerase chain reaction mRNA levels of P- and E-selectin, monocyte chemotactic protein-1, and IL-10. Groups were compared by analysis of variance and t tests. RESULTS: Less thrombus was noted at both days 2 and 6 in animals treated with cIL-10. At day 2 only, vein wall leukocyte counts revealed a significant decrease in neutrophils in cIL-10 animals versus controls, with no significant differences for vIL-10 animals. In cIL-10-treated animals, P-selectin protein levels were decreased at day 6, along with a decreased thrombus mass, without significant differences in E-selectin, monocyte chemotactic protein-1, or IL-10 protein levels. vIL-10 treated animals showed increased E-selectin mRNA and thrombus mass versus controls on day 6. CONCLUSIONS: cIL-10 is more antithrombotic/anti-inflammatory than vIL-10. This may be the result of cIL-10 decreasing P-selectin protein expression and vIL-10 increasing E-selectin mRNA levels.

Animals↗
PubMed2003

Neutropenia impairs venous thrombosis resolution in the rat.

OBJECTIVES: Neutrophil influx is one of the first events in a formed deep venous thrombosis (DVT), but whether these cells are active participants in the resolution process is not clear. This study tests the hypothesis that neutrophils (PMN) are active participants in DVT resolution. METHODS: Thrombosis was induced by inferior vena caval (IVC) ligation in male Sprague-Dawley rats, and rats were sacrificed at 2, 4, or 7 days for evaluation of the thrombus. Neutropenia was induced by rabbit anti-rat PMN serum, and controls received rabbit serum. Venography was performed at the 7-day time point. Immunohistochemical staining was performed to quantify intrathrombus PMNs and monocytes, and the myeloperoxidase (MPO) assay was performed to assess intrathrombus neutrophil activity. Intrathrombus concentrations of kerotinocyte cytokine (KC), macrophage inflammatory protein-2 (MIP-2), gamma interferon inducible protein-10 (IP-10), macrophage inflammatory protein-1 alpha (MIP-1 alpha), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-alpha were quantified by enzyme immunoassay at each time point and normalized to total protein. Total collagen was determined at day 7. RESULTS: Peripheral blood smears showed a 94% PMN reduction at 2 days (P <.05), recovering to 44% of control at 7 days. Intrathrombus PMNs were significantly lower in neutropenic rats at 2 and 4 days, but there were no differences in intrathrombus monocytes. The MPO assay confirmed reduced neutrophil activity at 4 days. Thrombi from neutropenic rats were larger at 2 and 7 days compared with controls. In vivo thrombus area at 7 days as assessed by venography was also greater in neutropenic rats as compared with controls. The intrathrombus KC concentration was increased more than 20-fold in the neutropenic rats at 2 days, but there were no significant differences in other intrathrombus chemokines. Finally, intrathrombus collagen was increased over threefold in neutropenic rats as compared with controls. CONCLUSION: Neutropenia impairs DVT resolution by several measures, most likely by altering normal fibrinolytic activity and thrombus collagen turnover.

Animals↗
PubMed2003

P-selectin and leukocyte microparticles are associated with venous thrombogenesis.

OBJECTIVES: P-selectin inhibition has been found to limit venous thrombosis. We hypothesize that elevated levels of P-selectin will amplify thrombosis, mediated by procoagulant microparticles (MPs). METHODS: Male mice (Mus musculus, n659), 20 to 25 grams, underwent IVC ligation to induce thrombosis. Groups consisted of wild type (WT) C57BL/6 controls, mice with high circulating levels of soluble P-selectin (CT), P-selectin gene-interrupted knockout mice (PKO), and E- and P-selectin gene-interrupted mice (EPKO). Additional groups were used to evaluate the ability of a P-sel antagonist (rPSGL-Ig) and an antibody directed against PSGL-1 to downregulate the effects of P-sel in CT mice and WT mice administered soluble P-sel at time of thrombosis. Animals were sacrificed on days 2 and 6 after IVC ligation. Thrombus mass (TM), vein wall morphometrics, and serum leukocyte/platelet microparticles (MPs) were evaluated by means of double-stained fluorescence-activated cell scanning analysis, and soluble P- and E-sel protein determination by ELISA. RESULTS: At days 2 and 6 in phase I of the experiment, significant differences (P <.01) in TM were noted between groups, with CT animals having the largest thrombi (50% and 57% increase in TM compared to WT at days 2 and 6) while EPKO mice had the smallest thrombi. Statistically, greater levels of neutrophils and total inflammatory cells were noted in the vein walls of CT animals at day 2 compared with WT and PKO animals. A significant difference was noted between CT and EPKO for neutrophils, monocytes, and total inflammatory cells, also at day 2. At day 6, the only statistically significant difference was found for monocytes, with a higher number in the CT animals than in WT animals. The evaluation of MPs revealed that the CT mice had a mixed leukocyte (MAC-1) and platelet (CD41) MP population that was also present in WT and PKO mice on day 2 and day 6. EPKO mice revealed a primarily platelet-derived MP population. Of interest, the CT mice with the highest TM showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs. CT mice revealed statistically higher levels of soluble P-selectin at days 2 and 6. In phase 2, an antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. CONCLUSIONS: This study demonstrates that high circulating levels of P-selectin are associated with increased thrombosis, whereas a lack of P-selectin and E-selectin is associated with a lessening of thrombosis. Additionally, leukocyte MPs are associated with venous thrombus formation. These data suggest the importance of selectins to venous thrombogenesis and show that P-selectin and leukocyte-derived MPs should be good targets to limit venous thrombus formation.

Animals↗
PubMed2003

P-selectin inhibition decreases post-thrombotic vein wall fibrosis in a rat model.

BACKGROUND: Post-deep vein thrombosis (DVT) venous insufficiency is a vexing problem despite effective anticoagulation, and is characterized by vein wall fibrosis. This study tested the hypothesis that P-selectin inhibition would decrease post-thrombotic vein wall fibrosis and associated profibrotic mediators. METHODS: A rat stasis model of DVT was used to produce a 2-day-old DVT. Rats then received either intravenous saline (control), rPSGL-Ig (4 mg/kg) once, or daily subcutaneous low molecular weight heparin (LMWH) (0.5 mg/kg). Inferior vena cava wall was harvested 7 days after treatment and processed for thrombus size; leukocyte content; profibrotic mediators by enzyme-linked immunosorbent assay; collagen I and III mRNA expression by semiquantitative real-time polymerase chain reaction; and for collagen protein. RESULTS: Thrombus mass and leukocyte counts were similar between the groups. Treatment with rPSGL-Ig and LMWH resulted in less vein wall collagen (P <.05). rPSGL-Ig treatment (and a similar trend for LMWH) was associated with decreased profibrotic mediators, including less IL-13, MCP-1, bFGH, and transforming growth factor-beta (P <.05). Collagen III gene expression, but not collagen I gene expression, was increased with LMWH treatment (P <.05). CONCLUSIONS: P-selectin inhibition with rPSGL-Ig or LMWH decreases post-DVT vein wall fibrosis, and is associated with decreased vein wall profibrotic mediators. This effect is independent of thrombus mass and vein wall leukocytes.

Animals↗
PubMed2003

Clinical application of sonographic elasticity imaging for aging of deep venous thrombosis: preliminary findings.

OBJECTIVE: Aging of deep venous thrombosis is an important and difficult clinical problem. Because it is known that thrombi harden as they mature, we have preliminarily tested sonographic elasticity imaging, a technique that estimates tissue hardness, to age venous thrombi. METHODS: Two adult patients with lower extremity thrombi were studied. One had a clinically chronic thrombus (at least 3 years old), whereas the other patient's thrombus was clinically subacute (25 days old). We performed freehand compression sonographic scans using a 5-MHz linear array transducer. Phase-sensitive B-scan frames were processed offline by a two-dimensional complex correlation-based adaptive speckle-tracking technique. The distribution of internal strains in the wall of the vein, thrombus, and surrounding tissue was analyzed. Clot hardness was normalized to the venous wall. RESULTS: The chronic clot was homogeneous, and the strain in the chronic clot was at least 10 times smaller than that in the vessel wall. The subacute clot was much more heterogeneous, and, on average, the strain magnitude in the clot was 3 to 4 times greater than that in the vessel wall. CONCLUSIONS: In this preliminary work, the 2 thrombi appeared very different, and these results suggest that elasticity imaging may be able to age deep venous thrombosis.

Adult↗