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 < 0.01). Addition of the free radical scavenger alpha-tocopherol had no effect on the ability of oxidized LDL to inhibit the Ca(2+)-ATPase. An increased cytoplasmic calcium level in whole platelets was induced by oxidized LDL (P < 0.01), indicating that the plasma membrane Ca(2+)-extrusion pump may also be inhibited in vivo by oxidized LDL, although other mechanisms for the increase in cytoplasmic calcium are possible. Since no change in membrane fluidity was observed in platelet plasma membranes exposed to oxidized or native LDL as estimated by steady state trimethylammonium diphenylhexatriene (TMA-DPH) anisotropy, oxidized LDL does not affect the Ca(2+)-ATPase by grossly changing the membrane environment. The present results suggest that exposure of platelets to oxidized LDL causes inhibition of the plasma membrane Ca(2+)-ATPase which contributes to the observed increase in cytoplasmic calcium and increased sensitivity to agonists.

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. At pH 7.4, the cationic charges of CFdmp exceeded those of CFah by a factor of 1.5 and the platelets bound approximately 1.5 times more CFah than CFdmp, suggesting the same number of anionic surface sites for both CF preparations. The capacity of the platelets to bind CF was diminished by 55% at 0 degree C or by 62% after aldehyde fixation and by 13% with PGE1. This suggests that the binding capacity depends on the mobility of the binding sites in the plane of the membrane but is only slightly increased by platelet activation. Binding to fixed or cold platelets approached equilibrium within a few seconds whereas saturation required several minutes at 37 degrees C. Neuraminidase preferentially reduced the slow binding and much less the rapid binding. Since activation by CF developed during seconds, suppressible by a brief treatment with neuraminidase 25 mU/ml, a small portion of neuraminidase-sensitive sites appears to be necessary for CF-induced platelet activation. Full activation and agglutination occurred at CF concentrations far below saturating concentrations. The results show that neither CF-induced activation nor agglutination depend on a simple neutralization of the negative surface charge.

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. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

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. The initially formed focal contacts with fibrin obviously persisted during clot formation. The physiological branching of the fibers enabled separation of platelets. The contact associated cytoskeleton formed a constricting and fiber internalizing sphere, but later stress fiber like bundles. As retraction progressed, the cytoskeleton changed to stress fiber connecting focal contacts with fibers. A model of clot formation in vitro is presented that reflects both the contributions of platelets (fibrin fiber internalization and retraction) and of fibers (branching) enabling the retraction.

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. Subendothelial structures, at the sites of those gaps, induce a formation of multilocal microthrombi of platelets. At some gaps within the endothelial layer a deposit of fibrin could also be observed hinting at a participation of the blood coagulation system in the sealing processes.

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. The morphological observations were supported by an analysis of the platelet shape-change parameter. Since the shape change, induced by oxidized LDL (50 micrograms/ml), was inhibited by a preincubation of platelets with staurosporine (10 nM), the protein kinase C was probably involved in the platelet activation initiated by oxidized LDL. The present results suggest that oxidized LDL could contribute to pathological thrombosis and atherogenesis by activating platelets.

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. The localization of the antiglycoprotein IIb-IIIa was time-dependent. After binding to the platelet surface membrane and open canalicular system, the surface-membrane labeling decreased during longer incubation periods. Preincubation with oxidized LDL inhibited the binding of antiglycoprotein IIb-IIIa. Our findings suggest that GP IIb-IIIa acts as a receptor for oxidized LDL. The binding of oxidized LDL to the GP IIb-IIIa might be the first step in platelet activation by plasma lipoproteins.

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 (0.03 U/ml) as compared to that which thrombin did alone (240 +/- 11 nM) (p < 0.05). The increased Ca2+ influx from the extracellular space is thought to be the reason of the increase in [Ca2+]i, since the effect of LDL was abolished by removal of external Ca2+ by EGTA. The DTS changed primarily from thin elongated forms to rounded vesicles. No evidence was noticed that LDL caused a mobilization of Ca2+ from the DTS.

Postnatal formation of the blood-testis barrier in the rat with special reference to the initiation of meiosis.

Postnatal formation of the Blood-Testis Barrier (BTB) in the rat was studied by either fixation in hypertonic fixative or employing lanthanum tracer. After 15 days of age, meiosis has reached different stages of spermatogenesis in different zones of the seminiferous cords. Only in those parts where germ cells are in the pachytene stage of meiosis do Sertoli cells form an effective barrier or tight compartment. Between 16 and 19 days of age, final formation of the BTB, which is to be found in the adult rat testis, occurs by zygotene and then leptotene stages successively entering the tight compartment. Thus, formation of a BTB by Sertoli cells does not occur synchronously along the length of the seminiferous cord but in accordance with the stage of meiosis of the associated germ cells.