Steven M. Burris

Inhibition of prostaglandin (PG) synthesis in sheep vesicular gland microsomes (SVGM) by nitroblue tetrazolium (NBT) and vitamin E (VE)

Previous studies have shown that NBT and VE together are potent inhibitors of platelet aggregation, secretion and PG synthesis. In this study, we evaluated the capacity of NBT to detect PG synthesis by SVGM. Aspirin pretreatment of SVGM decreased the amount of NBT reduced after addition of arachidonic acid, demonstrating that products generated by the cyclo-oxygenase were involved in NBT reduction. The influence of NBT and VE on PG synthesis by SVGM was then evaluated by measuring malondialdehyde (MDA) production. NBT or VE alone had no significant effect, but together these agents were as effective as aspirin in preventing MDA formation. The effect of NBT and VE on 14C-arachidonic acid conversion was followed by thin layer chromatography and radioscanning. Again, NBT or VE alone were ineffective, whereas the combination was as effective as aspirin in preventing conversion of arachidonic acid. We speculate NBT and VE together inhibit pg synthesis by scavenging a free radical species of arachidonic acid generated in the initial step of fatty acid peroxidation.

Influence of thrombin in suspension, surface activation, and high shear on platelet surface GPIb/IX distribution

Studies in our laboratory have shown that glycoprotein Ib/IX (GPIb/IX), the receptor for von Willebrand factor (vWf), is not decreased in number or cleared from exposed surfaces to the internal membranes of platelets activated in suspension by thrombin alone, by interaction with formvar surfaces alone, or by a combination of the two modes of stimulation. The present study has examined the influence of three different types of stimulation including activation by thrombin in suspension followed by surface activation on formvar, then exposure to high shear stress in a flat chamber. Samples were fixed for study in the electron microscope after each single stimulus, after the combination of two modes of activation, and after the combination of suspension, surface, and shear activation and were stained by an immunogold procedure using monoclonal antibodies to localize GPIb/IX on singly, doubly, or multiply activated cells. The results demonstrate that GPIb/IX receptors remain on activated platelets from edge to edge and that there is no difference in the number or distribution of receptor complexes on thrombin-activated platelets, surface-activated cells, or platelets exposed to the combination of suspension, surface, and shear activation. The findings add additional support to the concept that GPIb/IX is not a mobile receptor and is not cleared from exposed surfaces to internal membranes under physiologic conditions.

Cytoskeletons of X-linked GATA-1, G208S macrothrombocytes are deficient in talin.

An X-linked mutation in the GATA-1 transcription factor, G208S, causes macrothrombocytopenia and serious bleeding problems in affected male family members. The unique ultrastructural pathology of their platelets was described previously. The present investigation has evaluated the cytoskeletal proteins of the GATA-1, G208S macrothrombocytes of two male patients by page gel electrophoresis and Western blot analysis. The 235-245 KD cytoskeletal protein, Talin, was absent from their (PAGE) gels and undetectable by a specific talin antibody on Western blots.

Epinephrine-induced reversal of aspirin effects on platelet deformability

The present study has evaluated the influence of epinephrine on the resistance of platelets to aspiration into micropipettes, and the effect of epinephrine on the altered deformability of aspirin treated platelets. Unlike other platelet agonists previously studied, epinephrine stimulation did not alter platelet deformability at a concentration capable of causing platelet aggregation. Aspirin caused a dramatic decrease in the resistance of platelets to aspiration. Pretreatment of platelets with epinephrine prevented aspirin from altering platelet deformability, and exposure of platelets to epinephrine after treatment with aspirin reversed the increased deformability produced by the drug. Blockade of alpha-2 adrenergic receptors with yohimbine or clonidine prevented epinephrine antagonism of the mechanical effects of aspirin. The studies provide further evidence of the novel antagonism between epinephrine and aspirin on platelet structure and function.

GATA-1, G208S macrothrombocytes are deficient in talin: Immunofluorescence studies

Previous investigations from our laboratory identified the ultrastructural pathology and cytochemistry of macrothrombocytes (MTC) from patients with the X-linked, G208S varient of the GATA-1 mutation.A subsequent biochemical study of the MTC cytoskeletal proteins using polyacrylamide gel electrophoresis and western blot analysis revealed the MTC were deficient in the high-molecular weight, actin binding protein, talin. The present study has used immunofluorescent techniques to further characterize the talin deficiency. Results confirm that the GATA-1, G208S MTC are deficient in talin, and what little is present relocates to the undersurface of the plasma membrane following activations where it associates with adhesion plaques.

Influence of cytochalasin B (CB) on GP Ib distribution after thrombin or TRAP and before surface activation.

The receptor for von Willebrand factor (vWF) on human platelets, glycoprotein (GP) Ib/IX, has been shown in our studies to be an immobile complex when stimulated in suspension or on surfaces. Recent investigations have revealed that GP Ib/IX remains immobile on platelets activated in suspension followed by exposure to formvar surfaces that cause the cells to spread. However, since channels of the open canalicular system (OCS) are evaginated back on to the exposed surface during spreading, it was suggested that our study missed the clearance of GP Ib/IX from the exposed surface to internal membranes. The present study has added cytochalasin B after exposure of platelets to thrombin or TRAP in suspension in order to prevent spreading and movement of GP Ib/IX during subsequent exposure to surface activation on formvar grids. Results indicate that GP Ib/IX receptors remain randomly dispersed from edge to edge on platelets activated by thrombin or TRAP in suspension 10 minutes before treatment with CB followed by surface activation. Statistical analysis of the frequency of immunogold particles binding to monoclonal antibodies attached to GP Ib/IX revealed no significant reduction in frequency, translocation from cell edges or concentration of GP Ib/IX receptors in or around channels of the OCS. Results support the concept that GP Ib/IX is not cleared from exposed surfaces to the OCS of platelets activated by thrombin or TRAP and surface activation.

INFLUENCE OF THROMBIN TREATMENT IN SUSPENSION FOLLOWED BY ACTIVATION UNDER HIGH SHEAR ON BINDING OF vWF BY PLATELET GPIb. ▴ 967

INFLUENCE OF THROMBIN TREATMENT IN SUSPENSION FOLLOWED BY ACTIVATION UNDER HIGH SHEAR ON BINDING OF vWF BY PLATELET GPIb. ▴ 967