Biomedical subjects
Publications and source records attributed to J A Ware.
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Thromboxane A2 (TxA2) causes contraction of vascular smooth muscle and aggregation of platelets; paradoxically, it also induces formation of the vasodilator and antiaggregant prostacyclin by human endothelium. To determine if the molecular structure of the endothelial TxA2 receptor differs from that of the previously characterized receptor from placenta, we isolated a putative TxA2 receptor cDNA from a human endothelial library. The predicted amino acid sequence revealed a structure of 369 amino acids, in which a novel cytoplasmic tail replaced the carboxyl-terminal portion of the previously characterized TxA2 receptor; this divergence in cytoplasmic domains resulted from the nonsplicing of a potential intron in the placenta TxA2 receptor. Northern hybridization reveals that the expression of the TxA2 receptor in endothelial RNA decreases 6-fold following stimulation with an endoperoxide analog. Polymerase chain reaction using oligonucleotide primers specific to each cytoplasmic domain revealed that only the novel receptor was expressed in endothelium, while both receptors were expressed in placenta. Overexpression of the endothelial TxA2 receptor cDNA in Chinese hamster ovary cells conferred the ability to bind a known receptor antagonist and mobilize Ca2+ in response to TxA2 mimetics. This finding of a new TxA2 receptor in endothelium suggests that a family of these receptors may result from alternative splicing of the cytoplasmic (carboxyl) tail.
The kinetic mechanism of the human platelet S-adenosyl-L-methionine (AdoMet)-linked isoprenylated protein methyltransferase was studied and determined to be ordered bibi. AdoMet binds first, and S-adenosyl-L-homocysteine (AdoHcy) departs last. Simple N-acetylated farnesylated cysteine analogs, such as N-acetyl-S-farnesyl-L-cysteine (AFC), are excellent substrates for the enzyme. Although many N-acetylated farnesylated cysteine analogs are excellent substrates for the enzyme, analogs with bulky moieties adjacent to the farnesylcysteine are neither substrates nor inhibitors of the enzyme. Two molecules of this class, N-benzoyl-S-farnesyl-L-cysteine (BzFC) and N-pivaloyl-S-farnesyl-L-cysteine (PFC) are useful in sorting out the putative physiological role of the methyltransferase in mediating human platelet aggregation because their pharmacological activities are unlinked to methyltransferase inhibition. When studied as inhibitors of platelet aggregation, the analogs are as active, or more active, than bona fide methyltransferase inhibitors of similar structure. Therefore, although it is possible that methyltransferase inhibitors, such as AFC, inhibit the enzyme when applied to cells, the observed pharmacological effects appear to be unrelated to this blockade. The new FC analogs described here have revealed a new signal transduction target which will be of some interest to explore.
Inhibitors of the production of endogenous angiotensin II (A-II) can diminish the hyperplastic response produced by arterial injury in animals; however, a similar effect in humans has not been observed. To explain this discrepancy, we compared the effect of A-II on rat aortic smooth muscle cells (R-SMC) and smooth muscle cells derived from human saphenous veins (H-SMC). A-II (10-1000 nM) significantly increased the proliferative rate of R-SMC incubated in 10% serum, but a similar effect was not observed with H-SMC. Incubation of R-SMC for 24 hr with A-II (1 microM) produced a significant increase in cell size (7%) and protein production (18%), whereas no hypertrophic response was noted in H-SMC exposed to A-II. In neither R-SMC nor H-SMC did A-II, in any concentration, induce cell migration. Stimulation of R-SMC with A-II resulted in tyrosine phosphorylation of five proteins (approximately 120, approximately 108, approximately 68, 45, 42 kDa). The 42- and 45-kDa proteins, which we have previously identified as mitogen-activated protein kinases (MAP-K), remained phosphorylated for 1 hr. In H-SMC, only MAP kinases were tyrosine phosphorylated, but with less intensity than in R-SMC, and only for 20 min. In protein kinase C-depleted SMC, tyrosine phosphorylation of MAP kinase was inhibited in both cell types. A-II produced hypertrophy and hyperplasia of R-SMC, but not H-SMC. Differences in intracellular signaling might account for these disparate effects.(ABSTRACT TRUNCATED AT 250 WORDS)
At least seven bacteriophage lambda clones encoding structurally related but unique polypeptides with PKC activity have been isolated from mammalian brain, epidermis, and lung cDNA libraries. The possibility that additional isoenzymes are expressed in human blood platelets or megakaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing oligonucleotide primers corresponding to conserved peptide sequences. cDNAs encoding a novel PKC-related sequence, designated PKC-theta, and four (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia cells and platelets. PKC-theta lacks a conserved region (C2) that is present in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes. Significantly increased [3H] phorbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-independent PKC activity were found in COS cells expressing the transfected cDNA. Northern transfer analysis of mRNA from various human tissues revealed high level expression of PKC-theta in skeletal muscle, lung, and brain, and minimal expression in cardiac muscle, placenta, and liver. These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.
The tumor promoter phorbol myristate acetate (PMA) directly activates protein kinase C (PKC) and, in human platelets, induces aggregation, release of granular contents, mobilization of intracellular Ca2+ as detected by the photoprotein aequorin, and phosphorylation of the 47-kDa substrate (p47) of PKC. Whether PKC activation by PMA or other agonists requires translocation of PKC from the cytoplasm to the lipids of the platelet surface membrane, however, is not known. Bryostatin-1, a macrocyclic lactone that binds to PKC at or near the PMA-binding site and thus blocks PMA's proliferative effects, induced concentration-dependent p47 phosphorylation, platelet aggregation, release of dense granule contents, and a rise in cytoplasmic Ca2+ (ED50 = 2.0 nM) in intact aequorin-loaded platelets. Neither alpha-, beta-, delta-, or zeta-PKC isozymes translocated to the plasma membrane following bryostatin-1, although translocation of both alpha- and beta-PKC isozymes was seen in PMA-stimulated platelets. In a cell-free assay, bryostatin-1 activated PKC purified from platelets if the membrane-derived lipid phosphatidyl serine was present; however, if 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid or arachidonic acid, two lipid products formed in the cytoplasm of activated platelets, were present, no membrane-derived lipids were required for bryostatin-induced PKC activation. Thus, phosphorylation of PKC substrates and associated functional changes do not require isozyme translocation to the surface membrane, even when these events are induced by a direct activator of PKC.
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Desmopressin acetate (1-deamino-8-D-arginine vasopressin [DDAVP]) improves hemostasis in hemophilia A and von Willebrand's disease and in some platelet disorders. In complex cardiac operations, excluding simple coronary artery bypass graft procedures, we found that desmopressin reduced blood loss by 40% and the need for transfusion by 34%. Conflicting reports followed. Future trials should emphasize patients with excessive bleeding. A possible post-desmopressin prothrombotic state was studied after hip replacement surgery. The incidence of deep vein thrombosis associated with warfarin sodium therapy was the same as that associated with desmopressin plus warfarin therapy. No desmopressin-induced thrombotic tendency was detected. A trend toward reduced blood loss with desmopressin was not significant. During cardiac catheterization, the plasma von Willebrand factor level was correlated with hemodynamic variables, including pulmonary vascular resistance, pulmonary arterial pressure, and (inversely) with cardiac index. von Willebrand factor concentration was highest in mitral stenosis. The relationship of these factors to the response to desmopressin remains to be defined.
BACKGROUND: Cocaine ingestion has been associated with thrombosis of coronary as well as peripheral arteries, but the mechanism by which cocaine promotes thrombus formation is unknown. Accordingly, we determined whether cocaine activates human platelets by flow cytometric analysis of whole blood to which cocaine was added. METHODS AND RESULTS: Activated platelets were detected by "two-color" flow cytometric analysis of the binding of fluorescently labeled antibodies directed against either platelet-associated fibrinogen or P-selectin, which are found on the surface of platelets only after stimulation. Platelets were distinguished from other constituents of whole blood by their ability to bind an anti-glycoprotein Ib antibody bound to both activated and resting platelets. Incubation of whole blood with cocaine, in concentrations of 10 microM to 13 mM, induced significant increases in both platelet-associated fibrinogen (range of increase, 45 +/- 12% to 125 +/- 40%) and P-selectin expression (36 +/- 15% to 112 +/- 24%). In platelets suspended in either buffer or plasma, however, P-selectin expression was detected only at the highest cocaine concentration (85 +/- 13% increase in plasma and 59 +/- 7% in buffer). Neither aspirin nor the ADP scavenger apyrase inhibited cocaine-induced P-selectin expression. Cocaine inhibited the uptake of 14C-radiolabeled serotonin by platelets (IC50, 8.7 microM). P-selectin expression and fibrinogen binding were found after the addition of cocaine alone to blood taken from some but not all donors; however, platelet activation in response to submaximal concentrations of the agonists ADP or epinephrine was enhanced by a low concentration of cocaine added to blood from every donor. CONCLUSIONS: Cocaine, in concentrations similar to those found clinically, induces activation of individual platelets studied in whole blood from some but not all donors, and platelet response to physiological agonists is enhanced by cocaine. Thus, cocaine-induced platelet activation may contribute to thrombosis following cocaine ingestion.
Although functional receptors for thromboxane A2 and prostaglandin H2 (TxA2/PGH2) have been identified in platelets and vascular smooth muscle cells, receptor-mediated events in human endothelial cells stimulated by these endoperoxides have not been shown. Using cultured endothelial cells harvested from human umbilical or saphenous veins, we measured the effect of the TxA2 mimetic U46619 on mobilization of cytoplasmic calcium ([Ca2+]i), as well as release of prostacyclin and expression of the proto-oncogene c-fos, intracellular events that have been linked to [Ca2+]i rise in stimulated endothelial cells. Addition of U46619 to confluent fura 2-loaded endothelial cells caused a concentration-dependent rise in intracellular [Ca2+]i, with agonist concentrations of 300 nM producing a maximal [Ca2+]i rise. This [Ca2+]i rise was a uniform response observed in all individual endothelial cells throughout the monolayer, as shown by microspectrofluorimetric visualization. Similar effects were seen with a structurally dissimilar endoperoxide analogue, I-BOP, and with the naturally occurring endoperoxide PGH2. The initial [Ca2+]i rise was not reduced when extracellular [Ca2+]i was chelated with EGTA, but a later "plateau" phase was eliminated. An antagonist of the receptor for TxA2/PGH2 (SQ29548) strongly inhibited [Ca2+]i mobilization. Stimulation of endothelial cells with U46619 also transiently increased expression of the proto-oncogene c-fos, as determined by RNA hybridization, and induced a fivefold increase in prostacyclin release. Thus, endoperoxides can stimulate human venous endothelial cells by means of TxA2/PGH2 receptors, whose occupancy can activate intracellular events associated with functional changes.
We prospectively studied 712 consecutive patients during a 1-year period who presented with amnesia or loss of consciousness after nonpenetrating head trauma and who had a perfect Glasgow Coma Scale score of 15. Of the 67 (9.4%) patients with acute traumatic lesions disclosed by computed tomography (CT) of the head, 2 required neurosurgical intervention and 1 died. Four factors were statistically correlated (P < 0.05) with abnormal CT findings: Older age, white race, signs of basilar skull fracture, and being either a pedestrian hit by a motor vehicle or a victim of an assault. Sex, length of antero- or retrograde amnesia, forward and reverse digit spans, object recall, focal abnormality on the general neurological exam, and subjective complaints were not statistically correlated with CT abnormality. Using step-wise discriminant function analysis, no single item or combination of items could be used to classify 95% of the patients into either the normal or abnormal CT group. Therefore, regardless of age, mechanism of injury, or clinical findings, intracranial lesions cannot be completely excluded clinically on head-trauma patients who have loss of consciousness or amnesia, even if the Glasgow Coma Scale score is 15. However, only two patients (0.3%) required neurosurgical intervention.
BACKGROUND: Transforming growth factor-beta (TGF-beta) is released after vascular injury and may influence the healing response of the vessel wall. We investigated the effect of this growth factor on proliferation and migration of human venous smooth muscle cells (SMC) and the signal transduction mechanisms through which TGF-beta exerts this effect. METHODS: SMC derived from human saphenous vein were used in a 72-hour proliferation assay and a 6-day migration assay. Cells were exposed to TGF-beta alone and in the presence of platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), epidermal growth factor, and serum. The ability of TGF-beta to activate tyrosine kinases, phosphatases, or protein kinase C was evaluated by use of Western blotting with an antiphosphotyrosine antibody. RESULTS: In a concentration-dependent manner, TGF-beta inhibited proliferation induced by PDGF, b-FGF, epidermal growth factor, and serum. This inhibitory effect was independent of SMC density. TGF-beta also inhibited migration induced by PDGF and b-FGF. Exposure of cells to TGF-beta did not lead to tyrosine phosphorylation of cellular substrates or activation of protein kinase C. CONCLUSIONS: TGF-beta inhibits both migration and proliferation of human SMC. This inhibitory effect is not mediated through protein kinase C, mitogen-activated protein kinase, or tyrosine kinases.
The effect of several solvents on the in vitro activity of rat liver N-acetyltransferase (NAT) isozymes was examined. Dimethylsulfoxide (DMSO), dimethylformamide, dimethyl-acetamide, ethanol, and methanol all reduced NAT II activity in concentrations ranging from 1.25 to 10%. DMSO caused a similar magnitude of reduction in NAT I and NAT II activity. The inhibitory activity of DMSO was characterized as competitive in nature. These results indicate that solvents frequently used to solubilize arylamines may have significant inhibitory activity towards NAT in vitro.
The protein kinase inhibitor 1-(5'-isoquinolinesulfonyl)-2-methylpiperazine (H7) has been widely used because of its ability to inhibit cyclic AMP- and cyclic GMP-dependent protein kinases (PKA and PKG) and protein kinase C (PKC) at roughly equal concentrations; it is much less potent on other kinases. Previous studies in other laboratories have found that H7 samples from different commercial sources have different properties in cellular studies and protein kinase C inhibition assays. We now report the results of chemical and biological tests which show that H7 samples also differ in chemical structure, again depending on their commercial source. Chemical synthesis and NMR spectroscopy indicate that H7 from most suppliers has the structure originally proposed for H7, while "H7" from another supplier is in fact its 3-methylpiperazine positional isomer.
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To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.
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