A new model for in vitro clot formation that considers the mode of the fibrin(ogen) contacts to platelets and the arrangement of the platelet cytoskeleton.

The constitution of platelet-fibrin(ogen) contacts, the separation of the platelets initially aggregated, and the rearrangement of the platelet cytoskeleton during clot formation (0.5 to 60 minutes after thrombin stimulation) were investigated using ultrastructural and immunocytochemical techniques. After aggregation, fibrin polymerizing within focal contacts and from degranulating secretory granules contributed to the fibers.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers.

Vitamin E reduces platelet adhesion to human endothelial cells in vitro.

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol.

Oxidized low-density lipoprotein inhibits the binding of monoclonal antibody to platelet glycoprotein IIB-IIIA.

Previous studies have shown that oxidized low-density lipoprotein (LDL) induces platelet activation more effectively than native LDL. To achieve a better understanding of the mechanism underlying the activation of human platelets by oxidized LDL, the present study relates the effect of oxidized LDL to changes of binding characteristics for glycoprotein (GP) IIb-IIIa. Washed human platelets were treated by monoclonal antibody against GP IIb-IIIa, and the ligand-receptor complexes were revealed by immunocytochemical techniques on the ultrastructural level.

Vitamin E (alpha-tocopherol) does not inhibit platelet stimulation by oxidized low density lipoprotein in vitro.

Platelet-rich plasma were treated with increasing concentrations of vitamin E (alpha-tocopherol). Washed platelets were exposed to oxidized low density lipoprotein (LDL) and examined by aggregometry and electron microscopy. The treatment of washed platelets by oxidized LDL induced morphological signs of activation like pseudopodia formation and an increase in light transmission.

Oxidized LDL damages endothelial cell monolayer and promotes thrombocyte adhesion.

The influence of oxidized low density lipoprotein (LDL) on a human endothelial cell monolayer was examined. The resulting contraction of the oxidized LDL-damaged endothelial cells lets intercellular spaces become enlarged and therefore visible via light microscopy. Electron microscopy reveals that the structural damage facilitates thrombocyte adhesion and formation of microthrombi.

Oxidized low-density lipoprotein increases endothelial intracellular calcium and alters cytoskeletal f-actin distribution.

Central to the pathogenesis of atherosclerosis is an abnormally functioning endothelium and a consequent loss of vascular integrity. These abnormalities may be induced by haemodynamic factors, biochemical substances, and also by oxidatively modified low-density lipoprotein (LDL). To understand the mechanism by which oxidized LDL causes endothelial dysfunction, human umbilical vein endothelial cells (HUVECs) were loaded with FURA-2, and intracellular calcium mobilization was studied in acute (seconds after LDL was injected) or chronic (24 h after LDL was injected) preparations.

Influence of purified apoprotein E on platelet activation induced by serotonin.

Serotonin induces platelet activation. Purified apoprotein E of 300 micrograms/ml prevented morphological alterations of blood platelets stimulated with serotonin (5 microM). Lower concentrated apoprotein E showed no such clear effects.

Oxidized low density lipoprotein inhibits platelet plasma membrane Ca(2+)-ATPase.

Oxidized low density lipoprotein (LDL) has been shown to enhance platelet activation. Since platelet activation is accompanied by an increase in cytosolic calcium, the effects of oxidized LDL on plasma membrane Ca(2+)-ATPase, plasma membrane fluidity and cytoplasmic calcium were studied in human platelets and purified platelet plasma membranes. Our results demonstrate that oxidized LDL, but not native LDL, inhibits the activity of Ca(2+)-ATPase in purified platelet plasma membranes (P < 0.

High-density lipoprotein fails to inhibit serotonin-induced activation of blood platelets.

High-density lipoprotein (HDL) of 100-400 micrograms/ml did not prevent morphological alterations of human blood platelets treated with serotonin (1-5 microM). Highly concentrated HDL (1,200 micrograms/ml) appeared to activate platelets in vitro. These findings indicate that whole HDL may not inhibit agonist-induced platelet activation.

Endothelial cells injured by oxidized low density lipoprotein.

Cultured endothelial cells from bovine aorta were exposed to oxidized low density lipoprotein and examined by electron microscopy. The endothelial cells contracted slightly and the intercellular junctions became unclear. Some osmiophilic material increased in the cytoplasm.

Adhesion of washed blood platelets in vitro is advanced, accelerated, and enlarged by oxidized low-density lipoprotein.

In order to study the influence of oxidized low-density lipoprotein (Ox-LDL) on platelet functional morphology at an early activation stage, washed human blood platelets were stimulated by 100 micrograms/ml Ox-LDL at 37 degrees C. The settling and spreading process of stimulated and unstimulated platelets on Formvar-coated glass was observed for approximately 20 min by reflection contrast microscopy (RCM) and quantified by image analysis. Each group consisted of at least 250 platelets.

Outgrowing nerves in the foreleg of a mouse embryo as viewed by three-dimensional reconstruction from electron micrographs.

Electron micrographs from serial cross-sections of 12-day-old mouse forelegs were digitized and three-dimensional reconstruction of the data was carried out on an Apple Macintosh Quadra 700 computer using a program especially designed for this purpose. Two nerve endings of the palmar net of the median nerve were visualized together with their accompanying Schwann cells and the surrounding processes of fibroblasts. Naked axons invade straightly into the embryonic connective tissue and serve as contact guidance for the Schwann cells to follow.

Decreased adhesion of oxidized LDL-stimulated platelets caused by cytochalasin D.

The adhesion of human blood platelets is studied with an in vitro model using reflection contrast microscopy and an image analysis system. The adhesive feature is promoted by oxidatively modified low density lipoprotein, which also induces functional morphological changes of platelets. However, when washed platelets are pretreated with 0.

Oxidized LDL induces serotonin release from blood platelets.

Oxidized low-density lipoprotein (LDL) induces a release of serotonin from morphologically resting platelets and shape changed platelets. This suggests that oxidized LDL, a newly reported weak agonist, contributes to atherogenesis and thrombogenesis by stimulating platelets.

The Morphology of Thrombin-activated Platelets with Reference to Different Fibrinogen Concentrations as Revealed by Computer-aided Three-dimensional Reconstruction.

Washed human platelets incubated in different concentrations of fibrinogen were activated by thrombin and aggregated in the presence of Ca(2+) or did not aggregate when EDTA was present. They were analyzed by transmission electron microscopy and computer-aided three-dimensional reconstruction. The volumes and surface areas of the reconstructed models were calculated.

Functional morphological alterations of human blood platelets induced by oxidized low density lipoprotein.

The effect of oxidized low density lipoprotein (LDL) on the functional morphology of human platelets in vitro was studied by means of transmission electron microscopy. The washed platelets, stimulated by oxidized LDL (50-300 micrograms protein/ml), showed disc-sphere transformation, centralization of granules and complete degranulation in a dose- and time-dependent manner. A cytodamage in platelet membrane was induced by oxidized LDL leading to a lower electron density of cytoplasm compared to control.

Textured biomaterials as a model for studying formation of focal contacts and rearrangement of the contractile cytoskeleton in platelets.

Fibres of textured biomaterials (BM) enable platelets to adhere with formation of focal contacts. The contact structure and the reaction of the contact associated contractile cytoskeleton were studied using fibres of different flexibility/mobility: butyl-S-Sepharose (S), Polysulfone (PS) and Polyurethane (PU). Ultrastructural and immunocytochemical investigations were carried out to obtain information on the influence of tension on (1) the structure of the focal contacts; (2) the constricting cytoskeleton known to retract adherent collagen or fibrin fibres and (3) the cable-like bundles of actomyosin as observed in the clot.

Gold-labelled Low Density Lipoproteins Bind to Washed Human Platelets.

There is still disagreement about the presence of receptors for low density lipoproteins (LDL) in human platelets. Therefore, washed human platelets in suspension were incubated with gold-labelled LDL, and the binding sites for LDL were revealed by transmission electron microscopy on ultrathin sections and on surface replicas. The LDL-gold complexes bound to the platelet membrane and appeared in the open canalicular system.

Sertoli cell nuclear changes in human testicular biopsies as revealed by three dimensional reconstruction.

Serial semithin sections of human testicular biopsy material were used for three-dimensional reconstruction in order to obtain information about Sertoli cell nuclei in normal and pathologically altered seminiferous epithelia. The three dimensional reconstruction program is based on the triangulation of image point series. It includes a calculation modus for determining surfaces and an approximation formula for the estimation of volumes.

Influence of low density lipoproteins on cytosolic free Ca2+ concentration and dense tubular system in human platelets.

The cytosolic free Ca2+ concentration [Ca2+]i and dense tubular system (DTS) of washed human platelets were affected by low density lipoproteins (LDL) of 25 micrograms/ml at 37 degrees C for 10 minutes. After the incubation with LDL, the [Ca2+]i increased from 115 +/- 29 nM to 141 +/- 24 nM. LDL promoted the increase of [Ca2+]i (471 +/- 31 nM) induced by thrombin (0.

Effects of α-tocopherol (Vitamin E) on the Ultrastructure of Human Platelets In Vitro.

Washed human platelets were incubated with increasing concentrations of α-tocopherol. Spontaneous aggregation was induced by tocopherol (0.5 mM or above).

Low Concentration of LDL Enhances Platelet Reactivity In Vitro- a Morphological Study.

The isolated low density lipoprotein (LDL) has been shown to cause shape change, granule centralization and incomplete degranulation of human blood platelets at concentrations of 50 to 300 μg protein/ml in vitro. About half the number of platelets were discoid at the LDL concentration of 50 μg/ml. If the platelets were pretreated with LDL at a concentration of 100 μg/ml, aggregation could be induced by thrombin (0.

Effect of intravenously injected collagenase on the concentration of circulating platelets in rats.

An enzymatically induced irritation of the vessel wall by intravenous injection of collagenase (1.5 U per kg body weight) into rats resulted in a transient decrease of the platelet concentration in the flowing blood. This may be caused by an occurrence of inter-endothelial gaps distributed throughout the total area of the circulatory system, as could be observed by electron-microscopic studies.

Cationized ferritin as a platelet-stimulating surface probe. Binding to platelets and effects on platelet function.

Polycationic derivatives of ferritin containing primary amino groups (CFah) or tertiary amino groups (CFdmp) were potent platelet agonists inducing shape change, aggregation and secretion, but also agglutination in the presence of EDTA. Pretreatment of platelets with neuraminidase, PGE1, indomethacin, or creatine kinase/creatine phosphate inhibited CF-induced activation. In contrast, neuraminidase and PGE1 increased the agglutination by CF, indicating an inverse relationship between activation and CF-induced agglutination.