Effect of vitamin B6 vitamers on platelet aggregation

The vitamers of vitamin B₆ inhibit platelet aggregation. However, their effect is weak when used separately. After the supplementation of one vitamer, the concentrations of the other ones also increase in plasma due to the interconversion of vitamin B₆ forms. It can be suggested that different vitamers in blood can interact with each other. The aim of this work was to test the effect of different vitamer combinations on platelet aggregation in vitro. Platelet aggregation was induced by ADP, collagen or arachidonic acid and measured photometrically in a Chronolog aggregometer. The inhibition of platelet aggregation by the pairwise combinations of the vitamers was significantly stronger than that of each vitamer separately. The combinations of three and four vitamers were yet more effective, inhibiting platelet aggregation at the concentration of 4 μM. Possible involvement of inhibitory prostaglandins in the effect of vitamin B₆ was studied. The inhibition of platelet aggregation by the vitamers could be prevented by the antagonist of prostacyclin receptors, CAY10441 while the antagonist of prostaglandin D₂ receptors, MK 0524 was ineffective. The results suggest that vitamin B₆ vitamers cause a synergistic inhibitory effect on platelet aggregation at concentrations that can be mediated by the activation of prostacyclin receptors with prostaglandin E₁.     

The influence of platelet aggregation inhibitors on metastasis formation in mice (3LL)

Summary Platelets were suggested to play a specific role in the haematogenous spread of experimental tumours. To test this hypothesis mice were treated with various inhibitors of platelet function (acetyl-salicylic acid, RA 233, ben-cyclan-, cyproheptadine). The effect of treatment on the development of lung colonies after i.v. tumour cell injection as well as on the formation of spontaneous metastases from the solid Lewis-lung carcinoma was evaluated. A significant increase of lung colonies after pretreatment with the platelet aggregation inhibitors was found. The effect of long term treatment on spontaneous metastasis formation gave equivocal results. The present investigations do not support the importance of the integrity of platelet function as a prerequisite for metastasis formation.

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Der Einfluß von Plättchenaggregationshemmern auf die hämatogene Metastasierung bei Mäusen (3LL)

Zusammenfassung Von verschiedenen Autoren wurde eine spezifische Rolle der Blutplättchen bei der hämatogenen Metastasierung angenommen. Zur Prüfung dieser Hypothese wurden Mäuse mit verschiedenen thrombocytenaktiven Pharmaka behandelt (Azetylsalicylsäure, RA 233, Benzyclan und Cyproheptadin), und der Effekt dieser Behandlung sowohl auf die Ausbildung von Lungenkolonien nach i.v. Tumorzellinjektion als auch auf die Spontanmetastasierung des Lewis-lung-Carcinoms wurde gemessen. Die Behandlung mit Plättchenaggregationshemmern führte zu einer signifikanten Zunahme der Lungenkolonien, während kein wesentlicher Einfluß auf die Spontanmetastasierung nachweisbar war. Die vorliegenden Ergebnisse sprechen nicht für eine pathogenetische Bedeutung der Thrombocyten bei der experimentellen Metastasierung.

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Supported by a grant from the 'Deutsche Forschungsgemeinschaft (Hi 213/2).

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Parts of the present paper are submitted by H.H. to the Faculty of Medicine (Essen) as M.D. thesis.     

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

Platelet activation and thrombus formation are under the control of signaling systems that integrate cellular homeostasis with cytoskeletal dynamics. Here, we identify a role for the ribosome protein S6 kinase (S6K1) and its upstream regulator mTOR in the control of platelet activation and aggregate formation under shear flow. Platelet engagement of fibrinogen initiated a signaling cascade that triggered the activation of S6K1 and Rac1. Fibrinogen-induced S6K1 activation was abolished by inhibitors of Src kinases, but not Rac1 inhibitors, demonstrating that S6K1 acts upstream of Rac1. S6K1 and Rac1 interacted in a protein complex with the Rac1 GEF TIAM1 and colocalized with actin at the platelet lamellipodial edge, suggesting that S6K1 and Rac1 work together to drive platelet spreading. Pharmacologic inhibitors of mTOR and S6K1 blocked Rac1 activation and prevented platelet spreading on fibrinogen, but had no effect on Src or FAK kinase activation. mTOR inhibitors dramatically reduced collagen-induced platelet aggregation and promoted the destabilization of platelet aggregates formed under shear flow conditions. Together, these results reveal novel roles for S6K1 and mTOR in the regulation of Rac1 activity and provide insights into the relationship between the pharmacology of the mTOR system and the molecular mechanisms of platelet activation.     

Absence of platelet activating factor (PAF) mediated platelet aggregation: A new platelet defect

The failure of normal human platelets to aggregate in response to platelet activating factor (PAF) has not been previously observed. We report here the first case of a patient whose platelets did not aggregate to PAF on multiple occasions.     

The effect of aggregation and release on platelet prothrombin-converting activity.

Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel-filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.     

Bleeding disorder due to platelet prostaglandin H synthase-1 (PGHS-1) deficiency

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti-inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second-wave aggregation and propensity for disaggregation. Arachidonate-induced platelet aggregation was defective, whereas PGH₂-induced aggregation was normal. Platelet thromboxane A₂ (TXA₂) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB₂/3 10⁸ plt respectively (normal range was 49–81 ng/3 10 ⁸ plt), whereas they were normal in response to exogenous PGH₂, i.e. 71.4, 56.6 and 48.9 ng/3 10⁸ plts, respectively (normal range 49–85 ng/3 10⁸ plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS-1 and TXA₂ synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity-purified polyclonal antibodies highly specific for human PGHS-1 and TXAS, respectively. In two patients the 70 kD PGHS-1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS-1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS-1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS-1 protein which has an impaired catalytic activity.     

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition

Abstract

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08–0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC₅₀ of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC₅₀ of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.     

The effect of histamine H2 receptor antagonists on platelet aggregation in man

The in vivo and in vitro effects on the aggregatory response of human platelets to ADP and collagen of a series of imidazole and non-imidazole histamine H2 receptor antagonists, and imidazole derivatives, have been studied. Bolus i.v. administration of the antagonists cimetidine and oxmetidine was without effect. However, inhibition of platelet aggregation was observed in vitro with oxmetidine, metiamide and to a lesser extent burimamide, but not with cimetidine or the non-imidazole antagonist ranitidine. Of the imidazole derivatives only imidazole and its 1-methyl analogue significantly affected platelet aggregation. The relationship between potency as a histamine H2 receptor antagonist, the presence of an imidazole ring structure and the antiaggregatory effectiveness of these compounds is discussed. Although certain antagonists clearly inhibit platelet aggregation in vitro, effects are only seen at drug concentrations exceeding those achieved under normal therapeutic conditions; thus the clinical significance of these observations remains to be determined.