Novel Biomarkers Associated with Deep Venous Thrombosis: A Comprehensive Review

Primary and recurrent venous thromboembolic disease (VTE, deep venous thrombosis and pulmonary embolism) remain a significant source of morbidity and mortality in the hospitalized patient. Non-specific subjective complaints and lack of specific objective findings related to acute deep venous thrombosis (DVT) and pulmonary embolism (PE) complicate the diagnosis. There remains no single serum marker available to exclusively confirm the diagnosis of VTE. While D-dimer is highly sensitive and useful for diagnostic exclusion, it lacks the specificity necessary for diagnostic confirmation resulting in the need for a variety of additional studies (i.e.: duplex ultrasound, venography, V/Q scanning, helical thoracic and pelvic CT scans and pulmoary angiography). There is evolving research supporting the utility of various plasma markers as novel “biomarkers” for VTE including selectins, microparticles, interleukin-10 and other cytokines. This review attempts to examine recent literature assessing the utility of P-selectin, microparticles, D-dimer, E-selectin, thrombin, interleukins and fibrin monomers in the diagnosis and guidance of therapy for VTE

Decreased venous thrombosis with an oral inhibitor of P selectin

BackgroundP-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.MethodsMice 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.ResultsPSI-697 significantly decreased TW in WT and ^CT mice, with a treated vs nontreated TW of 132 ± 24 vs 228 ± 29 × 10−4 g (P = .014) and 166 ± 19 vs 281 ± 16 × 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.ConclusionsPSI-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 RelevanceDeep 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)

Direct evidence for tumor necrosis factor-alpha signaling in arteriogenesis

Background-Arteriogenesis serves as an efficient mechanism for flow restoration after arterial occlusion. This process is associated with inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), although their role in arteriogenesis remains unclear. We hypothesized that arteriogenesis is reduced in mice lacking functional TNF-alpha or p55 receptor. To test this hypothesis, we developed a novel microsphere-based murine model of hindlimb perfusion measurement. Methods and Results-Unilateral femoral arteries of nude (n=9), TNF-alpha(-/-) (n=9), TNF-alpha receptor p55(-/-) (n=8), and p75(-/-) (n=8) mice as well as their appropriate genetic back-round controls were occluded. The nude mice underwent laser Doppler hindlimb flux measurements preoperatively, postoperatively, and after 7 days. Seven days after ligation, all animals underwent tissue perfusion determinations using fluorescent microspheres. Laser Doppler findings confirmed acute decrease in flux with falsely normal values after I week. Microsphere results from control mice showed perfusion restoration to values approximate to50% of normal within 7 days. TNF-alpha(-/-) mice demonstrated a significant reduction (45.1%) in collateral artery perfusion compared with controls (TNF-alpha(-/-) 22.4+/-5.1% versus B6x129 49.7+/-9.3%; P <0.01). p55(-/-) mice exhibited an almost identical 45.8% reduction in collateral artery formation (p55(-/-) 28.3+/-4.3% versus C57BL/6J 61.8+/-9.1%; P <0.01), whereas p75(-/-) mice were equivalent to controls (p75(-/-) 54.5+/-5.5%; P=0.13). Conclusions-Microsphere techniques in mice offer a tool for the molecular dissection of arteriogenesis mechanisms. These results suggest that TNF-alpha positively modulates arteriogenesis probably via signaling through its p55 recepto

Arteriogenesis proceeds via ICAM-1/Mac-1- mediated mechanisms

Monocyte adhesion to shear stress-activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte adhesion via intercellular adhesion molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, monocyte chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or MCP-1 plus an IgG2a isotype control via osmotic minipumps. After 7 days, collateral conductance was evaluated: solvent 4.01 (mL/min per 100 mm Hg), MCP-1 plus ICAM-mab 8.04 (versus solvent P=NS), and MCP-1 alone 33.11 (versus solvent P <0.05). Furthermore, the right femoral arteries of ICAM-1-/-, Mac-1-/- and mice having defective selectin interactions (FT4/7-/-) as well as their corresponding controls were ligated. One week later, perfusion ratios were determined by the use of fluorescent microspheres. FT4/7-/- mice did not show any significant difference in perfusion restoration whereas ICAM-1-/- and Mac-1-/- mice had a significant reduction in arteriogenesis as compared with matching controls (FT4/7-WT 37+/-9%, FT4/7-/- 32+/-3%, P=0.31; C57BL/6J 59+/-9%, ICAM-1-/- 36+/-8%, P <0.05; Mac-1-/- 42+/-3%, P <0.05). ICAM-1/Mac-1-mediated monocyte adhesion to the endothelium of collateral arteries is an essential step for arteriogenesis, whereas this process can proceed via selectin interaction independent mechanisms. Furthermore, in vivo treatment with monoclonal antibodies against ICAM-1 totally abolishes the stimulatory effect of MCP-1 on collateral artery growth, suggesting that the mechanism of the MCP-1-induced arteriogenesis proceeds via the localization of monocytes rather than the action of the MCP-1 molecule itsel