Motivational interviewing training for medical students: A pilot pre-post feasibility study.

OBJECTIVE: To evaluate the impact of brief training in motivational interviewing (MI) from a non-specialist professional for medical students. METHODS: Students (n = 20) received three four-hour sessions of MI training over one week. They interviewed caregivers acting as patients in two standardised medical situations, six weeks before and three weeks after training. Global scores from the MITI-3.1.1 code, including "MI- Spirit", were attributed to the audiotaped interviews by two independent coders, blind the pre- or post-training status of the interview. Secondary outcomes were: caregivers' perception of students' empathy (CARE questionnaire), students' evaluation of self-efficacy to engage in a patient-centred relationship (SEPCQ score), and students' satisfaction with their own performance (analogue scale). RESULTS: MI-Spirit score increased significantly after training (p < 0.0001, effect size 1.5). Limited improvements in CARE score (p = 0.034, effect size 0.5) and one of the SEPCQ dimensions (sharing information and power with the patient; p = 0.047, effect size 0.5) were also noted. Students' satisfaction score was unaffected (p = 0.69). CONCLUSION: These findings suggest that brief MI training can improve communication skills in medical students. PRACTICE IMPLICATIONS: Such an intervention is feasible and could be generalised during medical studies.

Flavonoids purified from parsley inhibit human blood platelet aggregation and adhesion to collagen under flow.

INTRODUCTION: Blood platelets are directly involved in both haemostatic and pathologic thrombotic processes, through their adhesion, secretion and aggregation. In this study, we investigated the effect of genins (aglycone flavonoids without sugar group) isolated from parsley (Petroselinum crispum) leaves in vitro on human platelet aggregation and adhesion to a collagen-coated surface under physiologic flow conditions. MATERIALS AND METHODS: The aggregation and adhesion studies were monitored after pre-incubation of platelets with genins. RESULTS: Genins inhibited dose dependently aggregation induced by thrombin, ADP and collagen. The strongest effect was observed in collagen induced aggregation (IC50 = 0.08 ± 0.01 mg/ml). The HPLC identification of genins compounds revealed the presence of keampferol, apigenin and other not identified compounds. The aggregation tests showed that these compounds have anti-aggregating activity. In addition, adhesion of human platelets to collagen was greatly decreased (over 75 %) by genins (0.3 mg/ml). While the mechanism by which genins act is unclear, we suggest that these compounds may interfere with a multiple target step in the haemostasis process. CONCLUSION: These results show that genins isolated from parsley has a potent antiplatelet activity. It may be an important source of beneficial antiplatelet compounds that decrease thrombosis and cardiovascular diseases.

Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3.

Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)ß(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells.

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Breast tumor cells transendothelial migration induces endothelial cell anoikis through extracellular matrix degradation.

Mutual interactions between human breast cancer cells and endothelial cells were studied in a model mimicking tumor cell intravasation. MDA-MB-231 tumor cells and human umbilical vein endothelial cells (HUVEC) were cocultured on opposite sides of a Transwell filter allowing tumor cell contacts with the basement membrane of the HUVEC forming endothelium and tumor cell transendothelial migration. Confocal microscopy analysis showed that transmigrating MDA-MB-231 cells lay under the HUVEC, thereby inducing HUVEC detachment and tumor cell-HUVEC contact-dependent apoptosis. GM6001 a matrix metalloproteinase (MMP) inhibitor inhibited almost completely, the MDA-MB-231 cell transendothelial migration and the anoikis process. In this intravasation model, a tumor cell invasive mechanism was demonstrated (i) induction of extensive endothelial anoikis induced by degradation of the extracellular matrix (ECM) components, (ii) activation of pro-matrix metalloproteinase (MMP)-2 into MMP-2 by the MT1-MMP-TIMP (tissue inhibitor metalloproteinase) 2-pro-MMP-2 membrane complex and (iii) attraction and migration of metastatic cell through apoptotic endothelium. These interactions could partly explain the necrosis-angiogenesis relationship in tumor angiogenesis.

Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

Matrix metalloproteinases are involved in both type I (apoptosis) and type II (autophagy) cell death induced by sodium phenylacetate in MDA-MB-231 breast tumour cells.

The effects of sodium phenylacetate (NaPa), an antitumoral molecule, on cell death and matrix metalloproteinase (MMP) activities and synthesis were investigated in two metastatic breast tumour cell lines, MDA-MB-231 and MDA-MB-435, cultured on three-dimensional type I collagen gels (3-D cultures). In both cell lines, NaPa inhibited cell proliferation and induced apoptotic cell death as measured by TUNEL assay, with an IC(30) of 20 mM and 10 mM for MDA-MB-231 and MDA-MB-435 cells, respectively. In MDA-MB-231 cells, NaPa also induced (i) an autophagic process evidenced by the appearance of autophagic vacuoles and an increased phosphatase acid activity, (ii) the formation of pseudopodia and (iii) an increase in MMP-1 and MMP-9 secretion without affecting MT1-MMP. In NaPa-treated MDA-MB-435 cells, no autophagic vacuoles were formed but F-actin depolymerisation was observed. MMP-1, MMP-9 and MT1-MMP levels were strongly enhanced in these cells but MMPs were not secreted and accumulated intracellularly. When breast cancer cells were treated with NaPa in the presence of an MMP inhibitor (GM6001), apoptotic cell death decreased and the induction of autophagic vacuoles in MDA-MB-231 cells was inhibited. Taken together, these data suggest that MMPs are involved in the autophagic cell death and/or apoptosis of breast tumour cells.

Circular dichroism studies of type III collagen mimetic peptides with anti- or pro-aggregant activities on human platelets.

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The anti-thrombotic activity effects of different glycine mutations in this sequence were studied in regard with their different adopted conformations. The biological results could be correlated to the glycine propensity to adopt a more stable polyproline II helix conformation. The incorporation of these sequences in "collagen-like" alpha-triple-helix peptides shows a pro-thrombotic activity compared to a scrambled negative control peptide which possesses no significant activity.

Hemostatic effects of recombinant DisBa-01, a disintegrin from Bothrops alternatus.

A monomeric RGD-disintegrin was recently identified from a cDNA library from the venom gland of Bothrops alternatus. The corresponding 12 kDa-recombinant protein, DisBa-01, specifically interacted with alpha(v)beta3 integrin and displayed potent anti-metastatic and anti-angiogenic properties. Here, the interaction of DisBa-01 with platelet alphaIIb beta3 integrin and its effects on hemostasis and thrombosis were investigated. DisBa-01 bound to Chinese Hamster Ovary (CHO) cells expressing beta3 or alphaIIb beta3 and promoted their adhesion and the adhesion of resting platelets onto glass coverslips. The disintegrin inhibited the binding of FITC-fibrinogen and FITC-PAC-1 to ADP-stimulated platelets and inhibited ADP-, TRAP- and collagen-induced aggregation of murine, rabbit or human platelets. In a flow chamber assay, DisBa-01 inhibited and reverted platelet adhesion to immobilized fibrinogen. DisBa-01 inhibited the phosphorylation of FAK following platelet activation. The intravenous injection of DisBa-01 in C57Bl6/j mice, prolonged tail bleeding time as well as thrombotic occlusion time in mesenteric venules and arterioles following vessel injury with FeCl3. In conclusion, DisBa-01 antagonizes the platelet alphaIIb beta3 integrin and potently inhibits thrombosis.

A novel alpha(v)beta (3)-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis.

The integrin alpha(v)beta(3) is involved in multiple aspects of malignant cancer, including tumor angiogenesis and metastasis, which makes the receptor a key target for the development of anti-cancer therapies. We report here on the production, the characterization and the in vivo anti-angiogenic and anti-metastatic properties of a novel alpha(v)beta(3)-binding disintegrin, DisBa-01, isolated from a cDNA library made with RNAs from the venom gland of Bothrops alternatus. The 11,637 Da-recombinant monomeric form of DisBa-01 displayed an RGD motif and interacted with purified alpha(v)beta(3) integrin in surface plasmon resonance studies, in a dose-dependent and cation sensitive manner. A three-dimensional molecular model of DisBa-01 in complex with alpha(v)beta(3) predicted a large surface of contacts with the beta(3) subunit. DisBa-01 inhibited the adhesion of alpha(v)beta(3)-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC(50) = 555 nM and 225 nM, respectively), and transiently inhibited their proliferation without direct cell toxicity, but did not affect the binding nor the proliferation of a human breast cancer-derived cell line (MDA-MB-231) not expressing alpha(v)beta(3). In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC(50) = 83 nM). When injected intravenously to C57BL/6 mice together with B16F10 melanoma cells, DisBa-01 time- and dose-dependently inhibited lung metastasis monitored by bioluminescent imaging. We conclude that DisBa-01 is a potent new inhibitor of alpha(v)beta(3)-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes.

Syndecan-4 contributes to endothelial tubulogenesis through interactions with two motifs inside the pro-angiogenic N-terminal domain of thrombospondin-1.

Thrombospondin-1 (TSP-1) is an extracellular matrix protein that modulates focal adhesion in mammalian cells and exhibits dual roles in angiogenesis. In a previous work, we showed that a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1 (TSP18) induced tubulogenesis of human umbilical vein endothelial cells and protected them from apoptosis. Our results indicated that these effects were possibly mediated by syndecan-4 proteoglycan, since binding of TSP18 to endothelial extracts was inhibited by anti-syndecan-4 antibody. Syndecan-4 is a heparan-sulfate proteoglycan that regulates cell-matrix interactions and is the only member of its family present in focal adhesions. In this report, we demonstrate that a monoclonal antibody against syndecan-4 blocks TSP18-induced tubulogenesis. Furthermore, through 2D adhesion and 3D angiogenic assays, we demonstrate that two sequences, TSP Hep I and II, retain the major pro-angiogenic activity of TSP18. These TSP-1 motifs also compete with the fibronectin Hep II domain for binding to syndecan-4 on endothelial cell surface, indicating that they may exert their effects by interfering with the recognition of fibronectin by syndecan-4. Additionally, TSP18 and its derived peptides activate the PKC-dependent Akt-PKB signaling pathway. Blockage of PKC activation prevented HUVEC spreading when seeded on TSP18 fragment, and on TSP Hep I and TSP Hep II peptides, but not on gelatin-coated substrates. Our results identify syndecan-4 as a novel receptor for the N-terminus of TSP-1 and suggest that TSP-1 N-terminal pro-angiogenic activity is linked to its capacity of interfering with syndecan-4 functions in the course of cell adhesion.

[Bioluminescent imaging to monitor tumor progression and metastasis in live animal]. / L'imagerie par bioluminescence pour visualiser la progression tumorale et la métastase chez le petit animal.

Animal models allowing more sensitive and early detection of tumorigenesis and metastasis are instrumental in the fight for developing effective therapies against aggressive forms of cancer. In the present chapter, the advantages and limitations of the bioluminescent imaging (BLI) approach are discussed. Although BLI provides rapid, highly sensitive, noninvasive and quantitative detection of small tumors and micrometastases, several issues like the low anatomic resolution or the attenuation of the luminescent signal with tissue depth must be considered when using this technology.

Tissue engineering for full-thickness burns: a dermal substitute from bench to bedside.

Our aim was to obtain a viable and easily available dermal substitute (DS) for the definitive coverage of full-thickness burns. A DS composed of a collagen-glycosaminoglycan-chitosan dermal matrix (DM) colonized with foreskin fibroblasts (FF) is described. FF-colonized DS were compared to the DM seeded with adult dermal fibroblasts (DF). FF-colonized DS expressed more fibrillin and tropoelastin than that with DF. Reconstructed skin obtained with both FF- and DF-colonized DS similarly expressed laminin-5 and collagen VII at the dermal-epidermal junction. Both FF- and DF-colonized DS produced cutaneous wound healing mediators in a dose-dependent manner in the presence of platelet lysate. After freeze-thawing, the FF-colonized DS were recovered in culture and retained their ability to produce vascular endothelial growth factor. Grafting of DS into nude rats achieved a complete healing of a dermal-epidermal lesion with a good epidermalization.

Type III collagen mimetic peptides designed with anti- or pro-aggregant activities on human platelets.

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The alpha-triple helix peptides behave as type III collagen analogues supporting platelet aggregation, while the homotrimer which does not exhibit a triple-helical conformation inhibits type III collagen-induced human platelet aggregation. The incorporation of the octapeptide sequence in type III collagen mimetic peptides may lead to the loss of the anti-thrombotic activity for a pro-thrombotic one.

Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation.

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.

A soluble truncated cadherin induces breast cancer cell apoptosis and growth inhibition.

PURPOSE: To identify the characteristics and function of the truncated cadherin cDNA which encodes a soluble molecule containing the sequence of VE-cadherin extracellular domain repeats from repeat 1 to 4 (designated as CED1-4) and a secreting signal peptide at N terminal. METHODS: A pMSCV/CED1-4 vector was constructed. Recombinant retrovirus ReCED1-4 and ReEmpty were produced by 293 package cells and transfected into MDA-MB435 human breast cancer cells. The expression of CED1-4 in transfectants and their supernatant was analyzed by RT-PCR and Western blot, respectively. MDA-MB435 cell proliferation assays were performed in vitro and in vivo. CED-14-induced apoptosis was demonstrated using Annexin V binding, TUNEL and caspase 3 assays. The expression of integrin beta1 and c-fos mRNA was detected by RT-PCR. RESULTS: The constructed soluble CED1-4 encoded 484 amino acids and a secreting signal peptide (27 amino acids). CED1-4 was expressed by MDA-MB435/CED1-4 cells, and detected in the supernatant of CED1-4 tranfectants. CED1-4 transfection significantly inhibited the growth of MDA-MB435 cells in vitro and in vivo. About 22-fold increase in the early apoptotic cells in MDA-MB435/CED1-4 cells was observed as compared with MDA-MB435/empty cells. Increased activity of caspase 3 in MDA-MB435/CED1-4 cells was more than two times as compared with that of the control cells. Interestingly, integrin beta1 transcriptional level in MDA-MB435/CED1-4 cells was down-regulated as compared with control cells. The resistance of fibronectin to CED1-4 apoptotic inducibility was confirmed by detection of caspase 3. The blockage of c-fos transcriptional expression was detected in MDA-MB435/CED1-4 cells. CONCLUSIONS: The soluble truncated cadherin may be considered an apoptotic inducer and growth inhibitor in the MDA-MB435 breast carcinoma cell line. Down-regulation of integrin beta1 and blockage of c-fos expression may be related to CED1-4-induced apoptosis and growth inhibition.

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts).

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and alpha2beta1 integrin) for efficient platelet activation.

Antithrombotic effect of the type III collagen-related octapeptide (KOGEOGPK) in the mouse.

Platelet adhesion to subendothelial types I and III collagens exposed upon vascular injury plays a crucial role in hemostasis and thrombosis. We previously identified a KOGEOGPK sequence (O for hydroxyproline) within type III collagen interacting with platelets, and demonstrated a strong inhibitory effect of the KOGEOGPK peptide on human platelet interactions with type III collagen in vitro. In the present study, we tested the antithrombotic effect of KOGEOGPK in vivo. In a mouse model of pulmonary thromboembolism induced by intravenous injection of type III collagen and epinephrine, prior administration of 80 mg/kg KOGEOGPK reduced by 50% the size of thrombi embolized in lungs, compared to vehicle-treated mice (p<0.0001). In a mouse model of photochemically induced lesion of caecum venules and arterioles, intravenous injection of 80 mg/kg KOGEOGPK decreased by 76% the occurrence of arteriole occlusion 45 min after vascular injury (p<0.05). A moderate antithrombotic effect of KOGEOGPK was also observed in the injured venules. In addition, intracardiac injection of KOGEOGPK had no effect on the tail bleeding time. These findings demonstrate a substantial contribution of platelet interactions with the type III collagen-related KOGEOGPK sequence in thrombus formation in vivo with preferential involvement in arterial thrombosis.

Inhibition and reversal of platelet aggregation by alphaIIbbeta3 antagonists depends on the anticoagulant and flow conditions: differential effects of Abciximab and Lamifiban.

Shear influences platelet aggregate formation and stability, as well as the inhibitory capacities of antithrombotic drugs. We compared the inhibitory and disaggregating properties of two distinct alphaIIbbeta3 antagonists, Abciximab and Lamifiban, on platelet aggregation induced by adenosine diphosphate (ADP) (5 micromol/l) in platelet-rich plasma (PRP), in an aggregometer (poorly defined low shear, <100/s) and in a microcouette at arterial shear rate (1,000/s). Platelet aggregation was detected by changes in light transmission in the aggregometer (TA), and by particle counting with a flow cytometer (PA). Lamifiban (1 mumol/l) completely inhibited TA or PA induced by ADP in citrated PRP in the aggregometer or microcouette. In contrast, Abciximab (2 micromol/l) only partially inhibited PA in the microcouette while blocking both TA and PA in the aggregometer. Moreover, Abciximab did not reverse platelet aggregates formed either in the microcouette or in the aggregometer, whereas Lamifiban caused complete reversal. On the contrary, Abciximab completely inhibited platelet aggregation induced by ADP in hirudin/d-Phe-Pro-Arg-chloromethylketone PRP in the microcouette. Our results demonstrate a marked dependence of inhibitory capacity of Abciximab on shear conditions, with citrate anticoagulant responsible for the residual aggregation, in contrast to Lamifiban, another alphaIIbbeta3 antagonist interacting with a distinct site on beta3.

Amino-terminal fragment of urokinase inhibits tumor cell invasion in vitro and in vivo: respective contribution of the urokinase plasminogen activator receptor-dependent or -independent pathway.

The urokinase plasminogen activator (uPA) is implicated in both cancer cell invasion and angiogenesis. It can interact with a specific receptor (uPAR) via the epidermal growth factor (EGF)-like domain in the urokinase amino-terminal fragment (ATF) in a species-specific manner. Our previous studies showed that adenovirusmediated delivery of murine ATF (AdmATF) suppressed human tumor growth in mouse models, by inhibiting murine angiogenesis. However, we cannot exclude its putative inhibitory action on human cancer cell invasion through a uPAR-independent pathway. To further investigate the mechanisms of ATF, we constructed another adenovirus, AdhmATF, expressing humanized murine ATF (hmATF). hmATF binds to human uPAR but not to murine uPAR. We compared the antagonist effect of both AdmATF and AdhmATF on human and murine cancer cells. In vitro, the supernatant from AdhmATF-infected cells repressed 79% of membrane-associated uPA activity on human MDA-MB-231 cells, whereas that from AdmATF-infected cells repressed 35% of membrane-associated uPA activity. On murine LLC cells, the supernatant from AdhmATF-infected cells inhibited 29% of cell surface uPA activity, whereas that from AdmATF-infected cells inhibited 74% of cell surface uPA activity. Similar results were obtained in a cell invasion assay. In vivo, intratumoral injection of the adenoviruses into LLC tumors on day 24 postinjection induced lower but significant tumor growth suppression by AdhmATF (tumor volume was 1185 +/- 128 mm3), whereas suppression by AdmATF was greater (407 +/- 147 mm3). In the MDA-MB-231 tumor model, on day 52 postinjection, tumor size was 187 +/- 47 mm3 in the AdhmATF-treated group and 468 +/- 65 mm3 in the AdmATF-treated group. The LLC and MDA-MB- 231 cell lines transfected by mATF or hmATF genes showed growth inhibition In vivo equivalent to the results obtained by adenovirus treatment. These results demonstrate the strong anticancer activity of ATF even when its uPAR-binding affinity has been suppressed, and indicate that ATF exerts an antitumor effect via dual mechanisms: essentially through targeting the uPA-uPAR system via the EGF-like domain and partially through targeting a uPAR-independent interaction via the kringle domain.