Adhesive Properties Of Platelets Research Articles

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

The antioxidant and antiaggregant effects of the covalent bienzyme superoxide dismutase — Chondroitin sulfate — Catalase conjugate on platelets

A covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate (SOD-CHS-CAT) demonstrated the dose-dependent inhibitory action on induced platelet aggregation both in the presence and the absence of hydrogen peroxide. The antioxidant activity of SOD-CHS-CAT was noted at much lower doses than that of other CAT derivatives. The conjugate SOD-CHS-CAT inhibited platelet aggregation regardless of the nature of inducers; it also prevented platelet spreading on the glass surface. The latter suggests conjugate influence on adhesive properties of platelets. Novel properties of the SOD-CHS-CAT conjugate underlie prospects of its biopharmaceutical applicability as a tool for antioxidant therapy.

Adhesive Properties of Platelets from Different Animal Species

The use of large animals (e.g., pig and sheep) in human medicine, and the need to develop new therapeutic strategies for domestic animal diseases related to platelet disorders, require better characterization of the physiology of animal platelets. In this study, the ability of platelets from buffaloes, horses, pigs and sheep to adhere to immobilized autologous fibrinogen was compared with that of human platelets. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma (PRP) was obtained by centrifugation. Platelets, isolated by further centrifugation of PRP, were washed by gel-filtration on Sepharose-2B, counted and added to the wells of 96-well plates pre-coated with autologous fibrinogen. After different times of incubation, non-adherent platelets were removed, and the number of adherent platelets was assessed by measuring endogenous acid phosphatase activity. Horse platelets showed the strongest ability to adhere to autologous immobilized fibrinogen, being 1·7-, 3·1- and 2·3-fold more active than human, buffalo and porcine platelets, respectively. Sheep platelets were unable to adhere to autologous immobilized fibrinogen. Platelet activation by adenosine 5-diphosphate (ADP) increased both human and animal platelet adhesive response. ADP-stimulated sheep platelets were able to adhere to autologous immobilized fibrinogen, albeit to a lesser extent than platelets from the other animal species. The observed interspecies variability in adhesive properties of platelets may reflect structural differences, or differences in the availability of the fibrinogen receptor (glycoprotein IIb/IIIa) on the platelet surface.

Thrombin increases expression of fibronectin antigen on the platelet surface.

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

Studies on Aggregation and Adhesion of Platelets

SummaryA modification of the Wright method for the evaluation of the aggregation and adhesive properties of platelets is described; the test is relatively simple and can be performed in a clinical laboratory.Using this procedure, the characteristics of platelets acidified to a pH of 6.5 from their normal pH by addition of small amounts of acid citrate dextrose (ACD) solution to platelet-rich plasma were evaluated. Aggregation and adhesive properties were decreased but returned to normal when the acidified platelets were resuspended in nonacidified platelet-poor plasma. These results confirm the transfusion and isotopie labeling survival studies using acidified platelets and indicate no significant injury of the platelets as exhibited by their aggregation and adhesive characteristics which are considered to be indicative of hemostatic function.