Release of platelet activating factor (P.A.F.-acether) by isolated perfused rat kidney

Platelet-activating factor (a 1-0-alkyl-2-acetyl glyceryl-3-phosphorylcholine, P.A.F.-acether) causes the aggregation of platelets from various Mammalian species and the release of their granule content. P.A.F.-acether activity has been recovered in vitro from perfused isolated Rat kidneys, stimulated by the ionophore A 23187. The maximum release was reached 10 min. after addition of the ionophore. P.A.F.-acether from kidney exhibited the same physico-chemical and biological characteristics as P.A.F.-acether from leucocytes. These data demonstrate that the kidney secretes a mediator of immediate hypersensitivity (P.A.F.-acether) in the veinous vasculature. Therefore the kidney itself has the ability of inducing intravascular platelet aggregation with subsequent local increase in vasopermeability.     

Semi-synthesis and proposed structure of platelet-activating factor (P.A.F.): PAF-acether an alkyl ether analog of lysophosphatidylcholine]

We have studied the molecular structure of platelet-activating factor" (P.A.F.), a mediator of inflammation obtained from blood leukocytes, macrophages, and platelets themselves. We have semi-synthetized a substance that possesses all the known physicochemical and biological characteristics of P.A.F. from hog leukocytes. This was performed by successive methylation, hydrogenation, and acetylation of lysophosphatidylethanolamine plasmalogen. We therefore propose the following structure for P.A.F.: 1-0-alkyl-2-acetyl-glyceryl-3-phosphorylcholine. This molecular structure is not yet described among the numerous substances capable of inducing platelet aggregation and release.     

Der Einfluss von Papaverin auf Funktionen der Blutplättchen

Summary The adhesiveness and the ADP-induced aggregation of human blood platelets as well as the agglomeration and viscous metamorphosis initiated by thrombin was inhibited by papaverin. The release of biogenic amines and ATP from rabbit blood platelets induced by thrombin or other proteolytic enzymes was diminished. Also eupaverin and ethylpapaverin have an inhibitory effect on the platelet functions.     

Dissociation between inhibition of phospholipid methylation and production of PAF-acether by rabbit platelets

Platelet-activating-factor (PAF-acether, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is formed by and released from rabbit platelets stimulated with thrombin, with the ionophore A23187, with collagen and with the platelet-stimulating glycoprotein convulxin. We here show that 3-deazaadenosine (C3ado) and L-homocysteine (HCy), two inhibitors of platelet methylation, reduced the formation of PAF-acether and of its deacetylated product lyso-PAF-acether by rabbit platelets challenged with thrombin, under conditions where the accompanying aggregation was not significantly modified. In contrast, platelet aggregation induced by convulxin was completely and irreversibly blocked when C3ado and HCy were associated. Aggregation by thrombin was not affected by the methylation inhibitors even when ADP was scavenged and thromboxane formation was suppressed. Our results indicate that phospholipid methylation, thrombin-induced platelet aggregation and formation of PAF-acether can be dissociated. The formation of PAF-acether by rabbit platelets can be blocked by mechanisms other than inhibition of phospholipase A2, since the latter is not affected by C3ado and/or HCy.     

Effect of vasoactive amines on Weibel-Palade bodies in capillary endothelial cells

The presence and distribution of Weibel-Palade bodies in stomach and colonic mucosal microvessels after the administration of vasoactive amines (serotonin and histamine), the serotonin depletor reserpine, and the von Willebrand factor secretagogue thrombin, was studied by transmission electron microscopy. These agents elevated the number of Weibel-Palade bodies in all microvascular endothelial cells and especially in capillaries. It is concluded that vasoactive amines enhance the synthesis and secretion of large von Willebrand protein multimers by endothelial cells.     

Effect of vasoactive amines on Weibel-Palade bodies in capillary endothelial cells

The presence and distribution of Weibel-Palade bodies in stomach and colonic mucosal microvessels after the administration of vasoactive amines (serotonin and histamine), the serotonin depletor reserpine, and the von Willebrand factor secretagogue thrombin, was studied by transmission electron microscopy. These agents elevated the number of Weibel-Palade bodies in all microvascular endothelial cells and especially in capillaries. It is concluded that vasoactive amines enhance the synthesis and secretion of large von Willebrand protein multimers by endothelial cells.     

Der Einfluss von Calcium auf die Brenzcatechinaminfreisetzung

Summary (1) Experiments on isolated perfused suprarenals of cattle have shown that the acetylcholine-induced release of catechol amines, but not that of phenylethylamine, is dependent on calcium, since a perfusion with calcium-free Tyrode's solution abolishes the action of acetylcholine and not that of phenylethylamine. (2) Inincubation experiments with isolated chromaffin granules, calcium produces a dose-dependent, significant release of catechol amines even in physiological concentrations (2.5 mM). (3) Acetylcholine does not release catechol amines from isolated granules, either in the presence or in the absence of calcium.

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Forschungs-Stipendiat der A. von Humboldt-Stiftung.

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Early circulating erythroid progenitors (BFU-E) in sickle cell anemia

Sickle cell anemia (SS) patients can be divided into two sub-populations according to peripheral HbF levels. Patients with low (<9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (>9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity.More recently further heterogeneity has been found among these two groups. In LFSS patients GM-CSF is constitutively produced by unstimulated monocytes. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release an inhibitory factor(s). The nature of this factor has to be determined.In addition, there are distinct subpopulations of BFU-E responsiveness to growth factor (GM-CSF, IL-3): a) LFSS patients have a homogeneous BFU-E population, equally responsive to GM-CSF and IL-3; b) HFSS patients, in addition to this subpopulation, have a subset of BFU-E dependent exclusively on IL-3 which is 20 to 40% of the total number of circulating BFU-E. This is similar to BFU-E from normal individuals. Hence, LFSS BFU-E represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by at least constitutively produced GM-CSF and possibly other factors.These observations suggest a significant modification in BFU-E behavior in the subset of SS patients with low HbF levels and high hemopoietic stress. The heterogenous regulation of BFU-E in SS disease seems to be an epiphenomenon of HbF levels, and not vice-versa.     

Uptake of fat by fluorescent granular perithelial cells in cerebral cortex after administration of fat rich chow

Summary As reported previously, fluorescent granular perithelial cells (F.G.P.) are distributed along small blood vessels, possibly postcapillary venous vessels, in the cerebral cortex; these cells take up intraventricularly administered horseradish peroxidase efficiently. In this study it is shown that lipid substances of the blood are easily incorporated into F.G.P. and stored in their cytoplasm. The quantity of fat deposits in F.G.P. varies with the age of the animal and is very marked in old rats. The administration of elastase suppresses the fat uptake and/or facilitates the fat metabolism in F.G.P.Acknowledgment. This study was supported in part by a grant in aid for Scientific Research from the Ministry of Education of Japan (No. 448085). The authors also would like to express their hearty thanks to Eisai Co. for a supply of elastase.     

Human endoglin as a potential new partner involved in platelet–endothelium interactions

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng ^+/?) mice compared to Eng ^+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.     

Dissociation between inhibition of phospholipid methylation and production of PAF-acether by rabbit platelets

Summary Platelet-activating-factor (PAF-acether, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is formed by and released from rabbit platelets stimulated with thrombin, with the ionophore A23187, with collagen and with the platelet-stimulating glycoprotein convulxin. We here show that 3-deazaadenosine (C₃ado) and L-homocysteine (HCy), two inhibitors of platelet methylation, reduced the formation of PAF-acether and of its deacetylated product lyso-PAF-acether by rabbit platelets challenged with thrombin, under conditions where the accompanying aggregation was not significantly modified. In contrast, platelet aggregation induced by convulxin was completely and irreversibly blocked when C₃ado and HCy were associated. Aggregation by thrombin was not affected by the methylation inhibitors even when ADP was scavenged and rhromboxane formation was suppressed. Our results indicate that phospholipid methylation, thrombin-induced platelet aggregation and formation of PAF-acether can be dissociated. The formation of PAF-acether by rabbit platelets can be blocked by mechanisms other than inhibition of phospholipase A2, since the latter is not affected by C₃ado and/or HCy.     

The effect of piroxicam on platelet aggregation

Summary Piroxicam inhibited aggregation of human and dog platelets caused by collagen, but not by adenosine diphosphate (ADP). Release of platelet ADP was inhibited by piroxicam.     

The effect of piroxicam on platelet aggregation.

Piroxicam inhibited aggregation of human and dog platelets caused by collagen, but not by adenosine diphosphate (ADP). Release of platelet ADP was inhibited by piroxicam.     

Tyrosinase-cytalyzed conjugation of dopa with glutathione

Summary A convenient method is described for the preparation of 5-S- and 2-S-glutathionyldopa, based on tyrosinase oxidation of dopa in the presence of glutathione. The yields of 5-S, 2-S, and 6-S isomers produced were about 76, 12, and 5%, respectively.One of the authors (G.P.) thanks the C.N.R. (P.F. Chimica Fine e Secondaria) for partial financial support.     

Platelet aggregation and stimulation of leucocyte procoagulant activity by rickettsial lipopolysaccharides in rabbits and in man

Summary The effects in vitro of 4 purified lipopolysaccharide (LPS) preparations from Rickettsiae on platelets and leucocytes were studied in rabbits and in man. All LPS induced aggregation in rabbit platelet-rich plasma but to differing degrees. This activity was abolished by inactivation of complement. None of the preparations induced aggregation of human platelets. Both rabbit and human leucocytes, when incubated with each of the rickettsial LPS preparations, generated a potent procoagulant activity (tissue factor). These findings add further support to the concept that rickettsial LPS behave as typical LPS from gram-negative bacteria and may be relevant to the understanding of the mechanism(s) responsible for triggering intravascular coagulation in rickettsial diseases.     

Peptide-stimulated release of prolactin from the fowl anterior pituitary gland

Summary Anterior pituitary glands from broiler fowl were preincubated for 24 h in either medium 199 only or medium containing estradiol 17, following which they were incubated in medium containing thyrotrophin releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) or substance P (SP), alone or with the dopamine agonist, apomorphine. Estradiol priming stimulated release of prolactin and enhanced apomorphine-inhibition of prolactin release. TRH stimulated prolactin release, an effect reversed by apomorphine, and priming with estradiol potentiated both effects. VIP stimulated prolactin to a lesser degree and again this was inhibited by apomorphine and potentiated by estradiol. SP had little effect on the nonsteroid-primed pituitary, but stimulated release of prolactin after estradiol treatment, though less effectively than TRH or VIP.     

Cytotoxicity of mouse peritoneal cells after alloimmunization]

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.     

Serotonin reuptake inhibitors and cardiovascular diseases: a platelet connection

Selective serotonin reuptake inhibitors (SSRIs) are a heterogeneous group of new antidepressants that cause a well documented acquired but reversible serotonin deficiency in blood platelets. Platelets are small, anucleate cells and are the only blood cells specialized in storing peripheral serotonin. Platelets are also an integral part of the hemostatic process that is initiated during pathologic thrombus formation in cardiovascular diseases. Serotonin release from platelets is important for functional hemostasis as indicated by congenital diseases with serotonin-deficient platelets that can lead to life-threatening bleeding problems. The postulate that SSRIs should have an impact on cardiovascular diseases is therefore well founded. Cardiovascular effects of SSRIs have indeed been shown in a number of studies investigating the effect of SSRIs in patients with psychosomatic comorbidity. SSRIs reduce the incidence of recurrent myocardial infarction (MI) in patients suffering from post-MI depression. In addition, SSRIs inhibit tight clot formation of platelets in vitro, which points to a direct anti-thrombotic or pro-fibrinolytic effect of SSRIs.Received 16 June 2004; received after revision 9 September 2004; accepted 23 September 2004     

Effects of physalaemin, a vaso-active peptide from amphibian skin, on the excitability of an identifiable molluscan giant neurone of Achatina fulica Férussac.

We examined effects of several vasoactive peptides (substance P, physalaemin, neurotensin, bradykinin, angiotensin etc.) on the excitability of molluscan giant neurones identified in the subesophageal ganglia of Achatina fulica Férussac. Of these peptides, only physalaemin showed a remarkable excitatory effect on a giant tonically autoactive neurone.