Peter Wörner

Hyperreactivity by an enhancement of the arachidonate pathway of platelets treated with cholesterol-rich phospholipid-dispersions

Abstract Human-platelets were incubated at room temperature with washing solution (controls), cholesterol-free, or cholesterol-rich dipalmitoyl-lecithin dispersions. After washing, platelets from each of the three preparations exhibited a similar well-preserved discoid form. Only those platelets which were incubated for ≥ 120 minutes with cholesterol-rich lipid dispersions showed an increase in their reactivity to the aggregating agents epinephrine, ADP, hydrogen peroxide, arachidonic acid, thrombin, and A 23187. This effect required platelet activation high enough to evoke serotonin release. Shape change and primary aggregation were not altered by any of the pretreatments of platelets. Increased serotonin release was accompanied by an enhancement of the generation of thiobarbituric acid-chromogen. Indomethacin or acetylsalicylic acid suppressed the increase in serotonin release. This shows that the hyperreactivity depends on prostaglandin synthesis. The platelets treated with cholesterol-rich lipid dispersions can be used as an in vitro model for human platelets in various diseased states which are hyperre-active due to an enhanced prostaglandin synthesis.

Platelet aggregation and the release reaction induced by ionophores for divalent cations

Abstract A comparison is made of the effects of ionophores X-537A and A 23187 on platelets. In the presence of calcium, the release reaction promoted by A 23187 is enhanced; the platelets take up calcium and they clump. By contrast, X-537A induces a release reaction independent of external calcium and does not mediate calcium-uptake by platelets or induce a platelet aggregation. The effect of X-537A on platelets may not result from its property as a calcium-ionophore. The release reaction induced by A 23187 is most likely mediated by an increase in the concentration of cytoplasmic calcium ions.

Sequential effects of the thiol-oxidizing agent, diamide, on human platelets

Abstract Diamide elicits initial stimulative and delayed inhibitory effects on the aggregation of citrated platelet-rich plasma (PRP). Diamide (50 μM-5 mM) produces shape change and Ca 2+ -dependent aggregation, which both are inhibited by prostaglandin E 1 and cytochalasin B (60μM). Enhanced aggregation occurs when diamide is added to PRP simultaneously with ADP or collagen. The resulting aggregation, as well as aggregation caused by diamide alone, turns into disaggregation about 2 min after application of the aggregating agents. The increase of ADP aggregation is restricted to the primary wave. Release of 3 H-serotonin and secondary aggregation are inhibited by ≥ 10 μM diamide. Prior exposure of platelets to diamide (100 μM) leads to inhibition of collagen aggregation but not of ADP aggregation, though disaggregation occurs in both cases. The effects of diamide on platelet function may be mediated by lipid peroxides. Their accumulation is probably modulated by glutathione through glutathione peroxidase.

D-galactose-binding lectins induce a differential response of blood platelets

Abstract Platelets are strongly aggregated by 50 μg/ml of soybean agglutinin (SBA), Cytisus scoparius agglutinin (CSA I), and peanut agglutinin (PNA). The effects of SBA, CSA I, and PNA require pretreatment of the platelets with neuraminidase and are inhibited by D-galactose. PNA is the only one of these lectins which simultaneously induces the secretion and the concomitant shape change of platelets. Cytochalasin B enhances the effect of PNA but is inactive with SBA and CSA I. Thus the saccharide specificity of the lectins does not determine the kind of the platelet response for which additional binding properties of these lectins may be crucial.

Aggregation of activated platelets with Walker 256 carcinoma cells

ZusammenfassungWalker 256 Karzinomzellen bilden irreversible Aggregate mit Ratten-Thrombozyten, die durch ADP oder Serotonin aktiviert wurden. Serotonin induziert bei Thrombozyten die Formänderung, jedoch keine Aggregation. Der Aktivierungsgrad der Thrombozyten, der durch die Scheiben-Kugel-Transformation angezeigt wird, ist demnach für die Reaktion zwischen Thrombozyten und Walker-Zellen ausreichend. Dies wird durch Versuche mit sphäroiden gewaschenen Thrombozyten bestätigt: Diese Thrombozyten bilden ohne zusätzlichen Stimulus mit Walker-Zellen irreversible Aggregate. In vivo könnten Walker-Zellen mit Thrombozyten reagieren, die durch Wirbel im Blutstrom oder durch Kontakt mit Subendothel aktiviert wurden. Unsere Beobachtungen erklären, warum andere Autoren keine Reaktion zwischen Walker 256 Karzinomzellen und nicht-aktivierten Thrombozyten in vitro messen konnten, obwohl Thrombozyten bei diesem Tumor an der Ausbildung hämatogener Metastasen beteiligt sind.SummaryWalker 256 carcinoma cells form irreversible aggregates with rat platelets activated by ADP or serotonin. Since serotonin induces platelet shape change but not platelet aggregation the degree of activation indicated by the disc-sphere transformation is sufficient for platelets to interact with these tumor cells. This is confirmed by experiments with spheroid washed platelets which form irreversible mixed aggregates with Walker 256 carcinoma cells without a stimulus being required. This type of tumor cells could react with platelets in vivo, provided the platelets are activated by disturbed blood flow or contact with subendothelium. Our observations can explain why other authors found no interaction between Walker 256 carcinoma cells and non-activated platelets in vitro even though platelets contributed to the formation of blood-borne metastases of this tumor.

Aggregation and release reaction of washed platelets stimulated by lanthanum

Abstract Ionized lanthanum caused clumping of washed platelets. This clumping response could be reversed by chelating agents but was not impaired by known inhibitors of platelets aggregation. Aggregation by lanthanum was not restricted to the unique clumping properties of platelets but occurred in fixed platelets and red cells and was most likely based on an electrostatic interaction. Lanthanum was able to stimulate as well as to inhibit serotonin release from platelets. At a concentration of 1 mM, lanthanum evoked a release of serotonin from washed platelets at 37°C. This release reaction was inhibited at 18°C or by prior treatment of platelets with neuraminidase or NEM. At a high concentration (10 mM), lanthanum did not stimulate the platelet release reaction but inhibited that induced by all stimuli investigated, presumably due to a fixation of membrane molecules. The release reaction promoted by thrombin or A 23187, but not that by collagen, was inhibited by a low concentration of lanthanum (0.1 mM). This inhibition is based on an interaction of lanthanum with the stimuli rather than with the platelet surface.

The relationship between shape change and release reaction of lectin-stimulated platelets. Effects of cytochalasin B and prostaglandin E1

Abstract The turbidometric method indicates two phases of morphologic alterations (shape change) which accompany the release reaction induced by certain lectins. An initial increase in optical density (O.D.) is followed by a decrease in O.D. (second phase). The release of serotonin correlates with the second phase. To obtain the second phase of shape change and the concomitant release of serotonin higher lectin concentrations are necessary than those required for the initial shape change. Shape change and release proceed independently of external calcium. Cytochalasin B (CB) enhances the amplitude of the second phase and shortens the time elapsing between the addition of the lectin and the culmination of the shape-change curve. Simultaneously, the release of 3H-serotonin increases. CB also enables the second phase and the serotonin release to occur at lectin concentrations which per se produce only the initial change in O.D. Extent and kinetics of the initial phase are not stimulated by CB and are characteristic for the kind of lectin. Prostaglandin E1 or dibutyryl-cAMP inhibit both phases of the shape change and the release induced by lectins. This inhibition also affects the stimulation by CB. These findings suggest the involvement of a cAMP-sensitive step in lectin-induced initiation of platelet secretion. The initial shape change appears to reflect the initiation stage which is the prerequisite for the extrusion of platelet constituents. The transformation to the final extrusion phase requires either higher lectin concentrations or the presence of CB.

Selective release of serotonin from platelets by diffusion facilitated by the ionophore X-537A

Abstract X-537A and A 23187 are known to be ionophores for divalent cations. However, in contrast to A 23187, X-537A at a low concentration causes platelets to release serotonin but no adenine nucleotides or heparin-neutralizing activity (PF4). Nor does X-537A stimulate the procoagulant activity (PF3) of platelets. In addition, no uptake of calcium can be established in platelets treated with X-537A and no degranulation or typical granule fusion zones can be seen in electron micrographs of these platelets. Two phase partition studies demonstrate the transport of serotonin into an organic phase in the presence of X-537A. These results strongly indicate that X-537A selectively releases serotonin from platelets by transporting it through membranes. - At high concentrations of X-537A, a calcium-uptake by platelets with a concomitant release of PF4 and adenine nucleotides in addition to that of serotonin and an activation of PF3 can be established. - A comparison is made of the action of X-537A and A 23187, which induces the release reaction by transporting calcium through membranes.