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Biomedical subjects

A D Purdon

Publications and source records attributed to A D Purdon.

34 records · Page 2Linked to original sources
PubMed1987

Ionophore-stimulated rat basophilic leukemia cells produce PAF-acether.

Ionophore (A23187) stimulated rat basophilic leukemia (RBL-1) cells produce a lipid mediator which caused rabbit platelets to aggregate and which by using platelet-activating factor (PAF)-acether antagonists and high-pressure liquid chromotography was shown to be PAF-acether. Thus RBL-1 cells represent a cell type suitable for studying the coordinated release of three mediators of anaphylaxis: histamine, leukotrienes C4/D4 (slow-reacting substances) and PAF-acether.

Animals↗
PubMed1986

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s).

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme-linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

Animals↗
PubMed1985

Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets.

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.

Arachidonic Acid↗
PubMed1985

Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function.

We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.

Bradykinin↗
PubMed1985

Plasma kallikrein and prorenin in patients with hereditary angioedema.

Recent evidence indicates that plasma kallikrein is activated during acute attacks of hereditary angioedema. Plasma kallikrein is known to convert inactive renin, or prorenin, into an active proteolytic enzyme in plasma exposed to acid or low temperatures as well as in purified systems. To establish whether plasma kallikrein could activate prorenin under physiologic or pathologic conditions, prorenin to renin conversion was assessed at neutral pH in plasma deficient in C1 inhibitor (hereditary angioedema). In these plasma samples lacking the two major inhibitors of kallikrein and possessing less than 10% of the inhibitory activity of normal plasma, prorenin was not converted to an active enzyme despite conditions under which prekallikrein was completely activated to plasma kallikrein and despite normal prorenin concentrations and activability.

Adolescent↗
PubMed1984

Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation.

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.

Animals↗
PubMed1984

Regulation of the coagulant activity and surface binding of high molecular weight kininogen.

Cleavage of the procofactor, high molecular weight kininogen, by kallikrein, is required for expression of it cofactor activity. Coagulant activity associated with the light chain of high molecular weight kininogen is destroyed by proteolysis by factor XIa. The heavy chain of high molecular weight kininogen augments the binding of the light chain to activating surfaces without itself expressing coagulant activity.

Blood Coagulation Factors↗
PubMed1983

1-O-alkyl-linked phosphoglycerides of human platelets: distribution of arachidonate and other acyl residues in the ether-linked and diacyl species.

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l'-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l'-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-1'-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16:0, 18:0, 18:1 (delta 9), 18:2(n-6) and 20:4(n-6). The 1-O-alkyl and 1-O-alk-1'-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22:5(n-3) and 22:6(n-3) acyl chains. Arachidonate comprised 44% of the acyl residues in the sn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-1'-enyl-2-acyl species were 23% and 25%, respectively, based on all 20:4(n-6) being linked to the sn-2 position of all classes.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic Acid↗
PubMed1978

Isolation and identification of epithelial-like cells in culture by a collagenase-separation technique.

An operational criterion for the identification and isolation of epithelial-like (E) cells, based on their ability to cover and protect a collagen gel from the action of collagenase, has been developed. The E cells isolated by this collagenase-separation technique (CST) exhibited the ultrastructural features, including desmosomes and abundant tonofilaments, that are considered characteristic of this cell type. Unlike confluent cultures of fibroblast-like (F) cells, E cells were not found to have large external transformation-sensitive (LETS) protein on their surface membranes. The CST provides a nondestructive and efficient means of identifying and isolating E cells from mixed populations.

Cell Separation↗
PubMed1978

Kinetics of hydrolysis of dispersions of saturated phosphatidylcholines by Crotalus atrox phospholipase A2.

Dispersions of lamellar phase dipalmitoyl phosphatidylcholine (DDPC) and dimyristoyl phosphatidylcholine (DMPC) in 0.01 M CaCl2 were subjected to hydrolysis by phospholipase A2 (EC 3.1.1.4) from Crotalus atrox venom. The reaction was followed continuously by titrating the released fatty acids. For hydrolysis of gel phase phosphatides, the steady-state initial velocities were hyperbolic functions of bulk lipid concentrations. At the 'pre-transition' temperature (34 degrees C for DPPC, 15 degrees C for DMPC), there was a large increase in the Michaelis parameter Vmax but no change in the parameter Km. A model was devised to account for these observations, in which the enzyme desorbs from the lipid surface after hydrolysis. The desorption rate constant is postulated to increase above the pretransition temperature. For hydrolysis of liquid crystalline phosphatides, the reaction consisted of a short initial burst of hydrolysis, a long 'lag' period of very slow reaction, followed by a dramatic increase in the reaction rate. Addition of 10 mol% lysolecithin or fatty acid abolished the 'lag' period. It was postulated that the enzyme adsorbs irreversibly to the surface of the liquid crystalline phase. Reaction products are postulated to stimulate desorption of enzyme from the surface. Thus, temperature-dependent changes in the rate of hydrolysis of dispersed phosphatidylcholines are attributed to changes in the rate of desorption of the enzyme from the lipid surface.

Chemical Phenomena↗
PubMed1976

Detection of lipid phase transitions by surface tensiometry.

A technique for the detection of lipid phase transitions is described, which involves measurement of the surface tension as a function of temperature. In the case of insoluble lipids, such as dipalmitoylphosphatidylcholine (DPPC) the lipid is spread as a multibilayer film on an aqueous substrate, while in the case of water-soluble lipids such as lysophosphatidylcholine (LPC) the surface tension of aqueous sols is measured. Surface tension at the interface, is monitored using a Wilhelmy plate while the temperature is continuously varied. Discontinuities or changes in slope in the surface tension-temperature (gamma-T) curve reflect phase transitions in the lipid. In the case of DPPC, the technique has been used to demonstrate the well-known gel-liquid crystalline thermal transition. This occurs at 36-38 degrees C in the multibilayer films; in bulk DPPC-water dispersions the transition is at 41 degrees. Cholesterol has the effect of lowering the thermal transition and broadening the temperature range. In films containing DPPC-cholesterol at a molar ratio of 2:1 or less, the transition is not present. These results are in agreement with a large number of previous studies of this system. In the case of LPC sols, a phase transition at about 70 degrees was detected when the concentration of SPC was close to the critical micelle concentration (CMC) at 70 degrees. This transition appears to reflect an increase in the equilibrium constant for micelle formation at this temperature. At higher concentrations of LPC a transition at 30 degrees, corresponding to a gel-liquid crystalline transition, was also detected. A complete description of gamma as a function of concentration and temperature in the range 10(-7) to 10(-3) g cm-3 and 20 degrees to 80 degrees has been obtained for LPC sols. The CMC varies from 6 X 10(-6) g cm-3 at 20 degrees to 10(-5) g cm-3 at 80 degrees.

Binding Sites↗
PubMed1975

Lysolecithin and cholesterol interact stoichiometrically forming bimolecular lamellar structures in the presence of excess water, of lysolecithin or cholesterol.

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 degrees C unlike lysolecithin alone, which forms micelles, Thes results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggest another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin-cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.

Binding Sites↗
PubMed1975

Lysolecithin-cholesterol interaction. A spin-resonance and electron-micrographic study.

Another publication (rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O.(1975) Can. j. Biochem. 53, 189-195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r) and electronmicroscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol % cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol % cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-dosylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol %. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.

Binding Sites↗