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Biomedical subjects

E W Salzman

Publications and source records attributed to E W Salzman.

At least 19 recordsLinked to original sources
PubMed1992

Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.

Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.

Actins↗
PubMed1992

Heterogeneity in filamentous actin content among individual human blood platelets.

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.

Actins↗
PubMed1992

Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP.

Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.

Adenosine Diphosphate↗
PubMed1991

Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets.

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

Adenosine Diphosphate↗
PubMed1991

Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces.

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.

Adsorption↗
PubMed1991

Correlation of circulating von Willebrand factor levels with cardiovascular hemodynamics.

BACKGROUND: Valvular heart disease is associated with a decreased platelet circulating time and a thrombotic tendency. The possibility that these events are related to changes in von Willebrand factor (vWF), a multimeric glycoprotein released from endothelial cells and platelets that mediates platelet adhesion to the vascular subendothelium, has not been examined. METHODS AND RESULTS: We measured the vWF antigen (vWF:Ag) concentration in 43 patients undergoing cardiac catheterization for the evaluation of mitral (n = 17) or aortic (n = 10) stenosis or nonvalvular heart disease (n = 16). Mean vWF:Ag concentration was significantly higher in patients with mitral stenosis than in those without (212 +/- 84 versus 150 +/- 79 units/dl, p less than 0.02); this elevation was associated with a significant elevation of pulmonary vascular resistance (PVR) in the patients with mitral stenosis (186 +/- 49 versus 133 +/- 81 dynes-sec-cm-5, p less than 0.02). The vWF:Ag levels in the entire group of patients (regardless of the presence or type of valvular disease) varied directly with PVR (r = 0.72, p less than 0.0001) and with pulmonary artery pressure (r = 0.60, p less than 0.0001) and inversely with cardiac output (r = 0.64, p less than 0.0001). Changes in PVR, pulmonary artery pressure, or cardiac output could not be correlated with circulating levels of fibrinogen or beta-thromboglobulin, which may be released from activated platelets, nor with the endothelial cell product tissue plasminogen activator. CONCLUSIONS: The association of high vWF:Ag levels with increased PVR and decreased cardiac output in patients both with and without mitral stenosis suggests a hemodynamically induced increase in the endothelial release of vWF, which might contribute to a thrombotic tendency in these patients.

Cardiovascular System↗
PubMed1991

Low-molecular-weight heparinoid compared with warfarin for prophylaxis of deep-vein thrombosis in patients who are operated on for fracture of the hip. A prospective, randomized trial.

In a randomized, prospective trial, a low-molecular-weight heparinoid (Org 10172 [Lomoparan]) was compared with warfarin for efficacy and safety in preventing deep-vein thrombosis in 263 patients who had an operatively treated fracture of the hip. One group of patients received Org 10172 in a dose of 750 units subcutaneously every twelve hours until the ninth postoperative day; on the seventh postoperative day, warfarin was added to the regimen. The other group received only warfarin. Both drugs were begun preoperatively, immediately after the admission evaluation. In the patients who received warfarin, the desired prothrombin time was one and one-half times the control level. Deep-vein thrombosis was detected by 125I-fibrinogen scanning and impedance plethysmography and was confirmed by phlebography and compression ultrasonography. Deep-vein thrombosis was found in nine (7 per cent) of the 132 patients who received Org 10172 and in twenty-eight (21 per cent) of the 131 patients who received warfarin (p less than 0.001). Adverse reactions were not significantly different in the two groups. Major bleeding complications occurred in eight patients in the Org-10172 group, only four of whom were receiving the drug at the time of bleeding, and in five patients who were receiving warfarin (not significant). There was no difference in intraoperative loss of blood or in requirements for transfusion. We concluded that the low-molecular-weight heparinoid Org 10172 is a safe, convenient, effective antithrombotic agent for the prevention of venous thrombosis after an operation for fracture of the hip.

Aged↗
PubMed1990

Evidence that circulating 6keto prostaglandin E1 causes the platelet defect of Bartter's syndrome.

Bartter's syndrome is associated with activation of prostaglandin metabolism. In the present study we provide several lines of evidence that a circulating metabolite of prostacyclin, 6ketoPGE1 is responsible for a defect in platelet function present in patients with Bartter's syndrome. In platelet aggregometry studies, plasma from patients contained platelet inhibitory activity which was fully neutralized by coincubation with antibody directed against 6ketoPGE1. Fractionation of lipophilic extracts of plasma by high pressure liquid chromatography yielded a platelet inhibitory fraction which comigrated with authentic 6ketoPGE1 and was neutralized by anti 6ketoPGE1 antibody. Lastly, direct measure of the plasma concentration of 6ketoPGE1 by specific radioimmunoassay indicates a 2-fold increase in patients with Bartter's syndrome (133 +/- 9.1 vs 60.7 +/- 12.3 picograms/ml; p less than 025). These studies provide firm evidence that the platelet dysfunction present in patients with Bartter's syndrome is attributable to an increase in the plasma concentration of 6ketoPGE1. In addition, these data provide further evidence in support of the centrality of activation of prostaglandin metabolism in the pathophysiology of Bartter's syndrome.

Adult↗
PubMed1989

Activation of protein kinase C in platelets by epinephrine and A23187: correlation with fibrinogen binding.

Activation of protein kinase C (PKC), as revealed by phosphorylation of a 47 kd protein (p47), occurs in platelets stimulated by some agonists (eg, thrombin or phorbol esters). It is not known if activation of PKC occurs with pairs of agonists, such as epinephrine and A23187, that do not individually phosphorylate p47, nor is it known what role the concentration of cytoplasmic Ca++ ([Ca++]i) plays in these events. We stimulated aequorin-loaded platelets with subaggregating concentrations of epinephrine and A23187, neither of which by itself phosphorylated p47. The combination of agonists resulted in p47 phosphorylation, an increase in platelet-bound fibrinogen, and aggregation, but only if the concentration of each agonist was sufficient to increase [Ca++]i if it was added separately. Subaggregating concentrations of A23187 alone released platelet fibrinogen and increased platelet membrane binding of [3H]-phorbol dibutyrate, but these were not enhanced by epinephrine. Epinephrine and A23187 did not increase production of diacylglycerol. Thus, epinephrine and A23187 together activate PKC by a mechanism that does not require phospholipase C or enhanced binding of PKC to the plasma membrane; PKC activation in turn is correlated with enhanced platelet fibrinogen binding and aggregation. These events require an initial elevation of [Ca++]i above a threshold.

Blood Platelets↗
PubMed1989

Collagen banded fibril structure and the collagen-platelet reaction.

Bovine hide collagen dispersions were swollen in the pH range 1.6-7.0, treated with glutaraldehyde, and dialyzed to neutral pH. The intensity with which these collagens reacted with human platelets in plasma was studied by aggregometry and scanning electron microscopy. Collagen swollen at a pH below 4.25 +/- 0.30 and treated with glutaraldehyde exhibited greatly reduced platelet aggregating ability after restoration of neutral pH. In addition, the state of supramolecular order in these collagens was investigated by transmission electron microscopy and infrared spectroscopy. Native, insoluble collagen fibrils were found to lose their banded structure, as observed by transmission electron microscopy, reversibly when exposed to low ionic strength aqueous solutions below pH 4.25 +/- 0.30. During the disorder transition, which occurred by time dependent swelling of fibrils, but without their disaggregation, the packing order in the fibrils was largely abolished while the triple helical structure of individual collagen molecules was retained. Chemical modification of collagen by glutaraldehyde treatment was found to prevent recrystallization of collagen during dialysis to neutral pH but did not otherwise affect the collagen-platelet reaction. The results of altering collagen mass dose (concentration) demonstrated the critical importance of traces of banded fibrils which resisted disordering below pH 4.25. The data suggest that collagen preparations which are free of significant traces of banded fibrils, but which are made up of collagen molecules possessing triple helical structure do not induce platelet aggregation, irrespective of dose.

Animals↗