Synthesis of Sialyl LewisX Mimetics with E- and P-Selectin Binding Properties and Immunosuppressive Activity.

E- and P-selectins are adhesion proteins implicated in immune cell recruitment at sites of infection, making them important drug targets for diseases involving excessive and uncontrolled inflammation. In this study, we developed an efficient strategy to synthesize bicyclic galactopyranosides through a key stereoselective equatorial C4-propiolate addition and TMSCN axial C-glycosidation. The nitrile group can then be converted to the carboxyl and different bioisosteres at a late stage in the synthesis, allowing for various derivatizations to potentially enhance biological activity. The sialyl LewisX glycomimetic featuring this rigidified bicyclic galactopyranoside moiety prevents neutrophil adhesion to endothelial cells in vitro by binding to both E- and P-selectins. We show here that the axial carboxyl analogue blocks immune cell recruitment in vivo, demonstrating its potential as an immunomodulator.

The C-type lectin domain of CD62P (P-selectin) functions as an integrin ligand.

Recognition of integrins by CD62P has not been reported and this motivated a docking simulation using integrin αvß3 as a target. We predicted that the C-type lectin domain of CD62P functions as a potential integrin ligand and observed that it specifically bound to soluble ß3 and ß1 integrins. Known inhibitors of the interaction between CD62P-PSGL-1 did not suppress the binding, whereas the disintegrin domain of ADAM-15, a known integrin ligand, suppressed recognition by the lectin domain. Furthermore, an R16E/K17E mutation in the predicted integrin-binding interface located outside of the glycan-binding site within the lectin domain, strongly inhibited CD62P binding to integrins. In contrast, the E88D mutation that strongly disrupts glycan binding only slightly affected CD62P-integrin recognition, indicating that the glycan and integrin-binding sites are distinct. Notably, the lectin domain allosterically activated integrins by binding to the allosteric site 2. We conclude that CD62P-integrin binding may function to promote a diverse set of cell-cell adhesive interactions given that ß3 and ß1 integrins are more widely expressed than PSGL-1 that is limited to leukocytes.

A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells.

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Modifying ICCA with Trp-Phe-Phe to Enhance in vivo Activity and Form Nano-Medicine.

BACKGROUND: 1-(4-isopropylphenyl)-ß-carboline-3-carboxylic acid (ICCA) was modified by Trp-Phe-Phe to form 1-(4-isopropylphenyl)-ß-carboline-3-carbonyl-Trp-Phe-Phe (ICCA-WFF). PURPOSE: The object of preparing ICCA-WFF was to enhance the in vivo efficacy of ICCA, to explore the possible targeting action, and to visualize the nano-feature. METHODS: The advantages of ICCA-WFF over ICCA were demonstrated by a series of in vivo assays, such as anti-tumor assay, anti-arterial thrombosis assay, anti-venous thrombosis assay, P-selectin expression assay, and GPIIb/IIIa expression assay. The nano-features of ICCA-WFF were visualized by TEM, SEM and AFM images. The thrombus targeting and tumor-targeting actions were evidenced by FT-MS spectrum analysis. RESULTS: The minimal effective dose of ICCA-WFF slowing tumor growth and inhibiting thrombosis was 10-fold lower than that of ICCA. ICCA-WFF, but not ICCA, formed nano-particles capable of safe delivery in blood circulation. In vivo ICCA-WFF, but not ICCA, can target thrombus and tumor. In thrombus and tumor, ICCA-WFF released Trp-Phe-Phe and/or ICCA. CONCLUSION: Modifying ICCA with Trp-Phe-Phe successfully enhanced the anti-tumor activity, improved the anti-thrombotic action, formed nano-particles, targeted tumor tissue and thrombus, and provided an oligopeptide modification strategy for heterocyclic compounds.

Development of a Translatable Ultrasound Molecular Imaging Agent for Inflammation.

This study details the development, characterization and non-clinical efficacy of an ultrasound molecular imaging agent intended for molecular imaging of P-selectin in humans. A targeting ligand based on a recently discovered human selectin ligand was manufactured as fusion protein, and activity for human and mouse P- and E-selectin was evaluated by functional immunoassay. The targeting ligand was covalently conjugated to a lipophilic anchor inserted into a phospholipid microbubble shell. Three lots of the targeted microbubble drug product, TS-07-009, were produced, and assays for size distribution, zeta potential and morphology were established. The suitability of TS-07-009 as a molecular imaging agent was evaluated in vitro in a flow-based adhesion assay and in vivo using a canine model of transient myocardial ischemia. Selectivity for P-selectin over E-selectin was observed in both the human and murine systems. Contrast agent adhesion increased with P-selectin concentration in a dynamic adhesion assay. Significant contrast enhancement was observed on ultrasound imaging with TS-07-009 in post-ischemic canine myocardium at 30 or 90 min of re-perfusion. Negligible enhancement was observed in resting (no prior ischemia) hearts or with a control microbubble 90 min after ischemia. The microbubble contrast agent described here exhibits physiochemical properties and in vivo behavior suitable for development as a clinical imaging agent.

Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells.

Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Microwave Responsive Nanoplatform via P-Selectin Mediated Drug Delivery for Treatment of Hepatocellular Carcinoma with Distant Metastasis.

Hepatocellular carcinoma (HCC) with metastatic disease is associated with a low survival in clinical practice. Many curative options including liver resection, transplantation, and thermal ablation are effective in local but limited for patients with distant metastasis. In this study, the efficacy, specificity, and safety of P-selectin targeted delivery and microwave (MW) responsive drug release is investigated for development of HCC therapy. By encapsulating doxorubicin (DOX) and MW sensitizer (1-butyl-3-methylimidazolium-l-lactate, BML) into fucoidan conjugated liposomal nanoparticles (TBP@DOX), specific accumulation and prominent release of DOX in orthotopic HCC and lung metastasis are achieved with adjuvant MW exposure. This results in orthotopic HCC growth inhibition that is not only 1.95-fold higher than found for nontargeted BP@DOX and 1.6-fold higher than nonstimuli responsive TP@DOX but is also equivalent to treatment with free DOX at a 10-fold higher dose. Furthermore, the optimum anticancer efficacy against distant lung metastasis and effective prevention of widespread dissemination with a prolonged survival is described. In addition, no adverse metabolic events are identified using the TBP@DOX nanodelivery system despite these events being commonly observed with traditional DOX chemotherapy. Therefore, administering TBP@DOX with MW exposure could potentially enhance the therapeutic efficacy of thermal-chemotherapy of HCC, especially those in the advanced stages.

Association of SELP Polymorphisms with Soluble P-Selectin Levels and Vascular Risk in Patients with Type 2 Diabetes Mellitus: A Case-Control Study.

P-selectin, an adhesion molecule, is encoded by SELP and known as biomarker of endothelial as well as platelet dysfunction. SELP polymorphisms (rs6136, rs6127, and rs6125) and raised levels of soluble P-selectin (sP-selectin) have been associated with several disease conditions. The present study was aimed to determine the association of SELP variants and sP-selectin levels as well as vascular risk in Type 2 diabetes mellitus (T2DM) patients. The frequency of rs6136, rs6127, and rs6125 was assessed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). sP-selectin levels were measured using commercially available kits. Haplotypes were constructed using PHASE software. The data obtained from the above-said analyses was subjected to suitable statistical analyses. sP-selectin levels (ng/ml) were significantly higher in patients as compared to controls (p < 0.001). Out of total, 22% of patients were found to have very high vascular risk, 43.2% with high vascular risk, while 34.4% with moderate vascular risk. For both rs6136 and rs6127, frequency of variant allele was found to be significantly higher in patients as compared to controls and accounted for 2.4- and 1.5-fold risk of disease development, respectively. CAG was found to be associated with 4.5-fold risk towards disease development. In contrast, AGG was conferring the protective effect. Significantly high sP-levels were observed in patients with homozygous wild genotype of rs6136, all genotypes of rs6127, and heterozygous genotype of rs6125 as compared to respective controls. Significant difference was observed in P-selectin levels within moderate-risk category for rs6136. When compared between the categories, significant difference was observed for rs6136 and rs6127. Furthermore, patients with haplotypes AAA, AGA, and AGG were found to have significantly high sP-selectin levels as compared to controls. Significant difference in sP-selectin levels was observed within very high-risk as well as high-risk category. When compared between the categories, significant difference was observed for AGA and AGG haplotypes. The studied polymorphisms of SELP have shown significant association with sP-selectin levels as well as vascular risk in T2DM patients.

Mechanical properties of P-selectin PSGL-1 bonds.

The accurate determination of the mechanical properties of P-selectin and PSGL-1 is crucial for design and optimization of applications utilizing such bonds, e.g. biosensors and targeted drug delivery systems, as adhesion and mechanical interactions play a critical role in several key functions of biological cells. In current work, the spring constant and rupture force of a single P-selectin PSGL-1 ligand receptor bond and the Young's modulus of a layer made of these ligand receptors are reported. The work-of-adhesion of the P-selectin PSGL-1 interface is also characterized. In the reported experiments, PSGL-1 coated particles are deposited on a P-selectin coated substrate and their transient nanometer scale out-of-plane displacements are acquired employing a laser Doppler vibrometer as they are excited by an ultrasonic field. From the spectral response of a single particle, the resonance frequencies of its vibrational motion are identified, and with help of a particle adhesion model, the average rupture force and stiffness of a single P-selectin PSGL-1 ligand receptor are determined as Frupt = 171 ± 56 pN and kb = 0.56 ± 0.04 mN/m, respectively. Furthermore, the Young's modulus and work-of-adhesion of a layer of P-selectin PSGL-1 ligand receptors are extracted as E = 28.74 ± 3.96 MPa and WA = 70.0 ± 8.0 mJ/m2, respectively. Unlike Atomic Force Microscopy (AFM) and other probe-based techniques, the reported approach eliminates the need for direct contact with the sample, which could compromise the accuracy of the results by imposing unspecified additional contact interactions. Further, the current technique can be employed for measurements under various fluid flow conditions.

Platelets activated by the anti-ß2GPI/ß2GPI complex release microRNAs to inhibit migration and tube formation of human umbilical vein endothelial cells.

BACKGROUND: Patients with anti-ß2GPI antibodies display significantly higher platelet activation/aggregation and vascular endothelial cell damage. The mechanism underlying the correlation between platelet activation, vascular endothelial cell dysfunctions and anti-ß2GPI antibodies remains unknown. METHODS: In this study, we derived miR-96 and -26a from platelets activated by the anti-ß2GPI/ß2GPI complex and explored their role in modulating human umbilical vein endothelial cell (HUVEC) migration and tube formation. RESULTS: Anti-ß2GPI/ß2GPI complex induces the release of platelet-derived microparticles (p-MPs). The amounts of miR-96 and -26a in these p-MPs were also higher than for the control group. Co-incubation of HUVECs with p-MPs resulted in the transfer of miR-96 and -26a into HUVECs, where they inhibited migration and tube formation. The targeting role of these miRNAs was further validated by directly downregulating targeted selectin-P (SELP) and platelet-derived growth factor receptor alpha (PDGFRA) via luciferase activity assay. CONCLUSION: Our study suggests that miR-96 and -26a in p-MPs can inhibit HUVEC behavior by targeting SELP and PDGFRA.

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice.

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments.

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation.

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1ß, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs' inflammation, in a mouse model of acute inflammation.

Stiff substrates enhance monocytic cell capture through E-selectin but not P-selectin.

The stiffening of blood vessel walls is associated with inflammatory diseases, including atherosclerosis, diabetes, and obesity. These diseases are driven by the excessive recruitment of inflammatory leukocytes out of the bloodstream and into tissues, but whether vascular stiffening plays a direct role in this process is not clear. In this study, we investigated the possibility that leukocyte capture from blood flow is enhanced on stiffer substrates. We modeled blood flow in vitro by perfusing monocytic cells over hydrogels that matched the stiffness of healthy and diseased arteries. The hydrogels were coated with either E-selectin or P-selectin, which are the endothelial adhesion proteins known to mediate immune cell capture from flow. Interestingly, we discovered that cell attachment to P-selectin coated gels was not dependent on substrate stiffness, while attachment through E-selectin was enhanced on stiffer gels. Specifically we found that on E-selectin coated gels, cells attached in greater numbers, remained attached for longer time periods, and rolled more slowly on stiff gels than soft gels. These results suggest that vascular stiffening could promote leukocyte adhesion to vessel walls where E-selectin is expressed, but may have less of an effect when P-selectin is also present.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

Evaluation of the hemocompatibility and rapid hemostasis of (RADA)4 peptide-based hydrogels.

(RADA)4 peptides are promising biomaterials due to their high degree of hydration (<99.5% (w/v)), programmability at the molecular level, and their subsequent potential to respond to external stimuli. Interestingly, these peptides have also demonstrated the ability to cause rapid (∼15s) hemostasis when applied directly to wounds. General hemocompatibility of (RADA)4 nanofibers was investigated systematically using clot formation kinetics, C3a generation, and platelet activation (morphology and CD62P) studies. (RADA)4 nanofibers caused a rapid clot formation, but yielded a low platelet activation and low C3a activation. The study suggests that the rapid hemostasis observed when these materials are employed results principally from humoral coagulation, despite these materials having a net neutral charge and high hydration at physiological conditions. The observed rapid hemostasis may be induced due to the available nanofiber surface area within the hydrogel construct. In conclusion, our experiments strongly support further development of (RADA)4 peptide based biomaterials. STATEMENT OF SIGNIFICANCE: Biomedicine based applications of (RADA)4 peptides are being extensively studied for the purpose of improving drug carriers, and 3D peptide nanofiber scaffolds. However, this peptide's biocompatibility has not been investigated till now. One particular study has reported a revolutionary and very desirable ability of (RADA)4 peptide to achieve complete and rapid hemostasis, nevertheless, the literature remains inconclusive on the underlying molecular mechanism. In this manuscript we bridge these two main knowledge gaps by providing the much needed systematic biocompatibility analysis (morphology analysis, platelet and C3a activation) of the (RADA)4 based hydrogels, and also investigate the underlying hemostatic mechanism of this peptide-induced hemostasis. Our work not only provides the much-needed biocompatibility of the peptide for applicative research, but also explores the molecular mechanism of hemostasis, which will help us design novel biomaterials to achieve hemostasis.

Versatile multiple protein nanopatterning within a microfluidic channel for cell recruitment studies.

A novel approach combining self-assembly-based colloidal lithography and polydimethylsiloxane (PDMS) micromolding to generate complex protein nanopatterns for studying the mechanisms of leukocyte extravasation within microchannels is presented. Nanostructured surfaces sealed onto PDMS-molded microchannels are chemically functionalized in situ in an all-aqueous process to generate bi-functional chemical nanopatterns. Subsequent co-immobilization with proteins makes use of common non-covalent coupling (e.g. HIS-tags, FC-tags and biotin-tags), giving nanopatterns of arbitrary combinations of oriented, functional proteins. Up to three different proteins were simultaneously co-immobilized into the microchannel with nanoscale precision, demonstrating the complex patterns. As a proof-of-principle, a mimic of an inflamed endothelium was constructed using a macro- and nanoscale pattern of intercellular adhesion molecule 1 (ICAM1) and P-selectin, and the response of leukocytes through live cell imaging was measured. A clear result on the rolling behavior of the cells was observed with rolling limited to areas where ICAM1 and P-selectin are present. This micro/nano-interface will open new doors to investigations of how spatial distributions of proteins control cellular activity.

Effects of vessel size, cell sedimentation and haematocrit on the adhesion of leukocytes and platelets from flowing blood.

BACKGROUND: Leukocytes and platelets typically fulfil their functions through adhesion to the walls of vessels with different size, haematocrit and shear rate. OBJECTIVE: We aimed to investigate differential effects of these variables on leukocyte and platelet adhesion. METHODS: Blood with varying haematocrit was perfused at a range of wall shear rates through capillaries of depth 100 or 300 µm coated with P-selectin or collagen. RESULTS: Adhesion of leukocytes was much more efficient in the smaller capillaries, but was equal on the upper and lower surfaces and showed nearly identical shear rate dependence for either size of vessel. Platelets also adhered more efficiently in the smaller vessels (although the effect of size was not so great), and equally on upper and lower surfaces, but their adhesion was much less sensitive to increasing shear rate. In previous studies using vertically-orientated capillaries, leukocyte adhesion increased with increasing haematocrit (Am. J. Physiol.285 (2003), H229-H240). Here, in horizontal 100 µm capillaries, leukocyte adhesion was highly efficient at haematocrit of 10% but restricted to the lower surface. Adhesion decreased initially as haematocrit was increased to 30% and then increased slightly again at 40% haematocrit. Increasing haematocrit supported a monotonic increase in platelet adhesion in the horizontal capillaries. CONCLUSIONS: Platelets adhere efficiently over a wider range of sizes and shear rates, and at high haematocrit. Leukocytes adhere better in smaller vessels and at low haematocrit in horizontal vessels. The different behaviours may represent 'rheological adaptation' to functions in inflammation vs. haemostasis.

Holothurian glycosaminoglycan inhibits metastasis via inhibition of P-selectin in B16F10 melanoma cells.

P-selectin-mediated tumor cell adhesion to platelets is a well-established stage in the process of tumor metastasis. Through computerized structural analysis, we found a marine-derived polysaccharide, holothurian glycosaminoglycan (hGAG), behaved as a ligand-competitive inhibitor of P-selectin, indicating its potential to disrupt the binding of P-selectin to cell surface receptor and activation of downstream regulators of tumor cell migration. Our experimental data demonstrated that hGAG significantly inhibited P-selectin-mediated adhesion of tumor cells to platelets and tumor cell migration in vitro and reduced subsequent pulmonary metastasis in vivo. Furthermore, abrogation of the P-selectin-mediated adhesion of tumor cells led to down-regulation of protein levels of integrins, FAK and MMP-2/9 in B16F10 cells, which is a crucial molecular mechanism of hGAG to inhibit tumor metastasis. In conclusion, hGAG has emerged as a novel anti-cancer agent via blocking P-selectin-mediated malignant events of tumor metastasis.