In vitro enhancement of human platelet aggregation by somatostatin

Summary Very low concentrations of somatostatin (S-14) strongly potentiate the in vitro aggregation induced by collagen, ristocetin and arachidonic acid, but not that induced by ADP or epinephrine, in both human platelet rich plasmas and gel-filtered platelet preparations. Desensitization phenomena may be induced either by repeated addition of S-14 or long lasting contact between S-14 and platelets.     

In vitro enhancement of human platelet aggregation by somatostatin

Very low concentrations of somatostatin (S-14) strongly potentiate the in vitro aggregation induced by collagen, ristocetin and arachidonic acid, but not that induced by ADP or epinephrine, in both human platelet rich plasmas and gel-filtered platelet preparations. Desensitization phenomena may be induced either by repeated addition of S-14 or long lasting contact between S-14 and platelets.     

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.     

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.     

Guanylate cyclase in human platelets with different aggregability

The activity of human platelet guanylate cyclase, and the activation of the enzyme by sodium nitroprusside were decreased in platelets with increased aggregability; these platelets were obtained from diabetes mellitus patients. Anomalies in guanylate cyclase activity and ADP-induced aggregation were more pronounced in platelets from subjects with type II than those with type I diabetes.     

Guanylate cyclase in human platelets with different aggregability

Summary The activity of human platelet guanylate cyclase, and the activation of the enzyme by sodium nitroprusside were decreased in platelets with increased aggregability; these platelets were obtained from diabetes mellitus patients. Anomalies in guanylate cyclase activity and ADP-induced aggregation were more pronounced in platelets from subjects with type II than those with type I diabetes.     

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.     

Die lokale Leukozytenreaktion bei der intraperitonealen Infektion der Maus mitE. coli

Summary Intraperitoneal aggregation of leucocytes is produced in an identical manner by intraperitoneal application of different amounts of living and dead bacteria and bacterial products. The maximum accumulation occurs with medium dosages of bacteria or their products, with high dosages no leucocytic accumulation is produced. Pretreatment with selected LPS of bacteria enhances the intraperitoneal accumulation of leucocytes also with the high amounts of bacteria, whereas the reaction in not pretreated with LPS is suppressed. Parallel to the increase of the intraperitoneal leucocytic accumulation the animals are protected against the infection with high amounts of bacteria, to which they succumb if not pretreated with LPS.     

Macrophage origin of platelet activating factor]

Platelet-activating factor (P.A.F.) is a mediator of anaphylaxis released from human and Rabbit basophils which causes aggregation of platelets and release of their vasoactive amines. We have induced the release of P.A.F. from Rat peritoneal cells (P.C.) with ionophore A 23187. After fractionation of P.C. on 5-15% Ficoll gradients, P.A.F. was obtained from macrophage-rich but not from mastocyte-rich fractions and from adherent cells but not from non adherent cells. These data suggest an important new function for the macrophage: aggregation of platelets and release of their vasoactive amines and others mediators of inflammation.     

Die Wirkung des Hydrocortisons auf die infektionsbedingte lokale Leukozytenansammlung

Summary The aggregation of leucocytes provoked in the peritoneal cavity of the white mouse by intraperitoneal injection of livingE. coli is inhibited by large doses of hydrocortisone. At the same time, the animal's susceptibility to infection is enhanced. The aggregation of leucocytes induced by injection of a bacterial extract containing lipopolysaccharides is inhibited in a similar manner. The effect of hydrocortisone sets in rapidly and subsides within 24 h. The inhibitory effect of hydrocortisone on the lipopolysaccharid effect on leucocytes seems to be an important factor in the influence of hydrocortisone on the infectious process.     

Immunochemical study of protein anomalies in platelets of patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome]

Crossed immunoelectrophoresis of "Triton X-100" solubilized proteins from (125I) iodinated normal and abnormal (Bernard-Soulier and thrombasthenic) platelets was performed using rabbit antibodies raised against whole, normal platelets. In Bernard-Soulier platelets neither of the two surface oriented forms of GP I, glycocalicin and glycoprotein Ib (band 13) were detected. In two patients with type I thrombasthenia, the most prominent precipitate (No. 16) and platelet fibrinogen (No 24) were not detected whereas in one patient with type II thromblasthenia, fibrinogen was normally detected and the amount of protein 16 was around 15% of the normal level. The alloantibody IgG L..., able to induce a "thrombasthenic-like" reactivity in normal human platelets, has been shown to be directed against protein 16.     

An MD2-derived peptide promotes LPS aggregation,facilitates its internalization in THP-1 cells,and inhibits LPS-induced pro-inflammatory responses

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293T cells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.     

Exogenous fibronectin modifies the aggregation of collagen-stimulated human platelets

Fibronectins (FN) are adhesive glycoproteins whose role in platelet aggregation is unclear. Addition of 3, 6 and 12 micrograms/ml of human plasma FN in vitro to isolated human platelets, which had been freed from plasma FN by gel filtration and subsequently stimulated with collagen, inhibited the last stage of platelet aggregation. With 3 and 6 micrograms/ml of FN a shortening of the lag-time was also observed. These data showed that FN may play a role in platelet-collagen interaction as well as in platelet-platelet interaction.     

Platelet aggregation is inhibited by phycolectins

Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red algaHypnea japonica, inhibited adenosine diphosphate (ADP)-or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 g/ml of the lectin with, ADP (2 M) and collagen (0.2 g/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 g/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.     

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin) on cyclic nucleotide phosphodiesterases in human platelets

Summary The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.     

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylme tho xy coumarin) on cyclic nucleotide phosphodiesterases in human platelets

The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmeth oxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.     

Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopolysaccharide ofPorphyromonas gingivalis

Porphyromonas gingivalis 381 lipopolysaccharide (LPS) definitely exhibited mitogenic activity in purified B-cells, separated from spleens of LPS-responsive C3H/HeN mice and LPS-non-responsive C3H/HeJ mice by using a magnetic cell sorting system. The mitogenic activity induced byP. gingivalis LPS was incompletely inhibited by polymyxin B.P. gingivalis LPS also induced a higher production of interleukin-6 (IL-6) in splenic B-cells of C3H/HeN mice as compared withEscherichia coli LPS. Furthermore,P. gingivalis LPS, but notE. coli LPS, induced definite IL-6 production in C3H/HeJ mice.P. gingivalis LPS increased tyrosine, serine/threonine phosphorylation of proteins with various major induced bands in splenic B-cells of both C3H/HeN and C3H/HeJ mice. Additionally, radioiodinatedP. gingivalis LPS, similarly toE. coli LPS, bound to a 73-kDa protein on C3H/HeJ as well as C3H/HeN B-cells. ThusP. gingivalis LPS may activate B-cells of C3H/HeJ as well as C3H/HeN mice via the LPS-specific binding protein on the cells.     

Glycoprotein IIb/IIIa blockade inhibits platelet aminophospholipid exposure by potentiating translocase and attenuating scramblase activity

The present study investigated the mechanisms underlying the inhibition of platelet phosphatidylserine (PS) exposure by GPIIb/IIIa blockade. Platelet PS exposure induced by thrombin stimulation was cell-cell contact dependent. GPIIb/IIIa blockade by c7E3 or SR121566 inhibited thrombin-induced platelet PS exposure. Thrombin stimulation induced mild, while A23187 induced extensive platelet-derived microparticle (PDMP) generation. Thrombin-induced PDMP generation was not inhibited by GPIIb/IIIa blockade. Aminophospholipid translocase activity was reduced upon platelet activation by thrombin. The reduction of non-PS-exposing platelets was attenuated by GPIIb/IIIa blockade, while little translocase activity was seen in PS-exposing platelets. Thrombin increased scramblase activity slightly in non-PS-exposing platelets, which was inhibited by GPIIb/IIIa blockade, and markedly enhanced scramblase activity in PS-exposing platelets. Activation of platelet calpain and caspase-3 or cytosolic calcium mobilization were not altered by GPIIb/IIIa inhibition. Thus, GPIIb/IIIa blockade inhibits platelet PS exposure by enhancing translocase activity and attenuating scramblase activity, but does not inhibit PDMP generation. Received 13 December 2006; received after revision 5 February 2007; accepted 9 March 2007     

A gonadotropin releasing hormone analog induces spermiation in intact and hypophysectomized frogs, Rana esculenta

The sperm-releasing activity of a gonadotropin releasing hormone (GnRH) agonist, Buserelin (GnRH) and hypophysis homogenate (PD) preparations was studied in intact and hypophysectomized (PDX) frogs, Rana esculenta. In addition, human chorion gonadotropin (hCG) was tested in PDX animals, and GnRH antagonist (GnRHA) treatments were carried out in intact and PDX animals, in combination with the hormonal injections. GnRH or PD treatments were able to elicit spermiation in intact and PDX animals. While GnRH, injected 24 h later, was again effective in inducing spermiation in intact animals, this was not the case in PDX frogs. GnRHA counteracted GnRH effects in intact frogs. Moreover, in PDX animals GnRHA injections counteracted the sperm-releasing activity induced by hCG or GnRH, but failed to inhibit sperm-releasing activity induced by PD homogenate.