N. Savion

Induction of interactions between CD44 and hyaluronic acid by a short exposure of human T cells to diverse pro-inflammatory mediators.

Migration of T cells into extravascular sites of inflammation is mediated by cell–cell and cell–matrix adhesion receptors, including the hyaluronan‐binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short‐term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44‐dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500 s−1) conditions, of T cells to hyaluronan requires a T‐cell activation of 2–3 hr and is regulated by the cross‐linking of CD3, cytokines (e.g. interleukin‐2 and tumour necrosis factor‐α), and chemokines (e.g. MIP‐1β, interleukin‐8, and RANTES). This T‐cell adhesion was manifested by polarization, spreading and co‐localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan‐bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells.

Testing agonist-induced platelet aggregation by the Impact-R (Cone and plate(let) analyzer (CPA))

The Impact-R [Cone and plate(let) analyzer (CPA)] is useful to assess platelet adhesion in different diseases and to monitor antiplatelet therapy. The purpose of the present study was to adapt this system to test agonist-induced platelet aggregation. Blood samples were tested by light transmission platelet aggregometry (LTA), Impact-R regular test and Impact-R agonist-response test. In the latter, samples were pre-incubated for 1 min with an agonist leading to platelet activation, micro-aggregates formation and reduced adhesion. Impact-R regular test of ten healthy volunteers demonstrated platelet adhesion (surface coverage, SC) of 11.2 ± 2.6% while LTA induced by ADP, ristocetin, epinephrine, collagen and arachidonic acid (AA) yielded maximal aggregation (81% to 93%). In the Impact-R agonist-response test, SC was reduced to 2.2 ± 1.0%, 1.2 ± 0.9%, 2.3 ± 1.0%, 2.2 ± 0.8% and 2.4 ± 0.4%, respectively. Prostaglandin E1 treatment weakened SC reduction in response to ADP and epinephrine (SC of 8.8 ± 1.8% and 9.5 ± 2.0%, respectively). Inhibition of P2Y12 receptor with 2MeSAMP resulted in a dose-dependent decrease in maximal aggregation in the ADP-induced test, which inversely correlated to SC in the Impact-R ADP-response test. The Impact-R agonist-response tests detected aggregation defects in patients with storage pool disease, severe von Willebrand disease and epinephrine response deficiency, and may be useful to assess the effect of different agonists on platelet aggregation.

Differential response of platelets to chemokines: RANTES non‐competitively inhibits stimulatory effect of SDF‐1α

Summary.  Background: Among the chemokines related to CXC and CC receptor groups and released from platelets, leukocytes and endothelial cells, SDF‐1, TARC and MDC have been found to be platelet agonists. Platelets do not contain SDF‐1α. In contrast, RANTES is constitutively present in platelet α‐granules and released upon platelet activation. Objectives: To study a possible role of RANTES as a modulator of SDF‐1α effect on platelets, in relation to CXCR4 and various CC receptors. Methods: CXCR‐4 (CXCL12) receptor expression and platelet activation were evaluated by flow cytometry, platelet deposition was studied by cone and plate(let) analyzer, and platelet aggregation by turbidometric aggregometry. Results: Flow cytometry studies revealed similar expression of CXCR‐4, the specific receptor of SDF‐1α on intact, inactivated, and activated platelets. Preincubation of platelets with RANTES affected neither CXCR‐4 expression, nor SDF‐1α binding to the platelet membrane. In the presence of fibrinogen, SDF‐1α activated gel‐filtered platelets. RANTES did not activate platelets, but substantially (by 70%) inhibited SDF‐1α‐induced fibrinogen binding. Similarly, RANTES abrogated the promoting effect of SDF‐1α on whole blood platelet adhesion to endothelial cell monolayer under venous flow conditions. In platelet‐rich plasma, RANTES moderately inhibited SDF‐1α‐induced platelet aggregation, while it did not affect aggregation induced by thrombin‐receptor activation peptide, adenosine diphosphate, or phorbol 12‐myristate 13‐acetate. A synergistic inhibitory effect of RANTES and prostaglandin E1 used at subthreshold concentrations, on SDF‐1α‐induced aggregation and SDF‐1α‐induced fibrinogen binding to platelets was observed, which may suggest involvement of RANTES in a cAMP‐dependent signal transduction pathway. Conclusions: RANTES non‐competitively inhibits activation of platelets by SDF‐1α, and thus may play a regulatory role in platelet response to inflammation.

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP

The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s-1) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s -1 ) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.

Activated platelets mediate Staphylococcus aureus deposition on subendothelial extracellular matrix: the role of glycoprotein Ib

Extracellular matrix (ECM) derived from bovine corneal endothelial cells was used as a model to study the role of platelets in Staphylococcus aureus interaction with the vascular subendothelium. In whole blood, S. aureus activated platelets, as demonstrated by P-selectin expression on the platelet membrane. Subjecting platelet-rich plasma (PRP) to the ECM under oscillatory conditions resulted in platelet adhesion and aggregation. S. aureus increased platelet deposition on ECM depending on the bacterium-platelet ratio. Scanning electron microscopy revealed that S. aureus adhered to and formed clusters on ECM-bound platelets. These findings were confirmed by using [3H]thymidine-labeled bacteria that adhered to the surface more extensively after deposition of platelets on ECM. Platelet pre-treatment with prostaglandin E1 resulted in inhibition of bacterial adherence. Glycoprotein (GP)Ib was involved in the bacterium-platelet interaction, as indicated by the following: (i) S. aureus diminished the binding of GPIb but not of GPIX or GPIIb-IIIa monoclonal antibodies (Mab) to washed fixed platelets; (ii) GPIb Mab inhibited S. aureus -induced platelet aggregation in a dose-dependent fashion; (iii) blockade of von Willebrand factor (vWf) binding to GPIb by a recombinant vWf fragment reversed the enhanced platelet deposition on ECM in the presence of S. aureus but did not affect platelet deposition in the absence of bacteria. The results indicate that S. aureus activates platelets and promotes their deposition on ECM via GPIb-dependent mechanism and that adherent platelets mediate S. aureus deposition on the subendothelium. These interactions might play a role in the pathogenesis of bacterial endocarditis.

Cellular Functions Related to Metastasis Differing between Low- and High-Malignancy Variants of AKR Lymphoma

Two AKR lymphoma variants of low (LM) and high (HM) malignancy were characterized for cell properties important for metastatic capacity. Previous data suggested that these murine lymphoma variants differed in the late phase of metastasis. In the present study we attempted to determine more accurately the stage in which they differ. The HM cells attached less efficiently than the LM ones to both endothelial cells (5 times) and extracellular matrix (twice), possibly indicating a more efficient extravasation capacity of the HM cells. The level of heparinase was low in the two variant cells. Attachment to liver sections was, however, more efficient with the HM than with the LM cells.

Effect of He−Ne laser on platelet activation and aggregation

Irradiation of whole blood with He−Ne laser inhibits platelet adhesion and aggregation on extracellular matrix under conditions of high shear rate and reduces fibrinogen binding and expression of P-selectin on the plasma membrane induced by a thrombin analog.

Metastasis-related cell functions in primary and metastatic tumor cells of AKR lymphoma.

The metastatic phenotype is of extreme complexity. To complete all the stages of metastasis, the tumor cell must possess a whole series of functional abilities. Multiple biological markers are therefore needed to achieve a deeper understanding of the metastatic phenotype. In the present study we compared primary (PT) to metastatic tumor (MT) cells of two AKR lymphoma variants with respect to several cellular functions relevant to various steps of tumor dissemination. The MT cells of the TAU-44 variant had a higher capacity than the PT cells to attach to endothelial monolayers and ECM, exhibited a more elevated motility and a higher capacity to grow in the spleen as a metastatic target organ. However, the TAU-44-MT cells had a lower ability to grow in the kidney than the PT cells. The TAU-33-MT cells had a higher ability to attach to endothelial cells and to grow in both spleen and kidney but were less motile compared to PT cells. Metastatic cells showed, on the whole, higher ability to perform in most, but not all, stage-specific models than primary tumor cells.

Integrilin Dose Optimization Using Cone Plate Analyzer – What Have We Learned Thus Far

Objectives: To assess platelet inhibition by cone and plate analyzer (CPA) in patients with acute coronary syndromes (ACS) receiving eptifibatide (Integrilin). Background: With the current use of glycoprotein IIb/IIIa (GP-IIb/IIIa) inhibitors during ACS and percutaneous coronary interventions (PCI), the need to measure platelet activity and optimize dosing became apparent. CPA is a novel technique to assess platelet activity in high shear stress conditions, mimicking arterial flow. Method: 40 consecutive patients with ACS received eptifibatide [2 intravenous boluses (180 µg/kg each) spaced 10 min apart, and subsequently a maintenance drip (2 µg/kg/min)]. All patients received aspirin, clopidogrel and unfractionated heparin or enoxaparin in conventional doses. Blood was obtained at baseline, 30 min and 3 h, after eptifibatide initiation, and was sent out for CPA core lab, blinded to patient therapy, characteristics, and sampling time. Results: At 30 min and 3 h only 45 and 60% of patients, respectively, reached GP-IIb/IIIa blockade of ≧95%. 25 and 15%, respectively, had <85% GP-IIb/IIIa blockade. No demographic, clinical, or laboratory predictors for eptifibatide resistance could be identified. Conclusion: <85% GP-IIb/IIIa blockade occurs in a high proportion of patients with ACS treated with eptifibatide. The clinical significance of this phenomenon, and the optimal way to assess and treat it warrant additional research. It is essential, however, to measure platelet activity or GP-IIb/IIIa occupancy in response to therapy, since no demographic, clinical, and laboratory predictors of eptifibatide refractoriness can be identified at this time at the bedside.