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T C Detwiler

Publications and source records attributed to T C Detwiler.

At least 37 records · Page 2Linked to original sources
PubMed1986

Thiol-disulfide exchange by thrombospondin: evidence for a thiol and a disulfide bond protected by calcium.

Thrombospondin (Tsp), a protein secreted by activated platelets, forms disulfide-linked complexes with thrombin [K. J. Danishefsky, R. J. Alexander and T. C. Detwiler (1984) Biochemistry 23, 4984]. Thiols and disulfide bonds of Tsp were analyzed, and a search was made for other Tsp covalent complexes. Platelets in 1 mM EDTA were activated with ionophore A23187, and the secreted proteins were analyzed by gel electrophoresis in sodium dodecyl sulfate. One millimolar dithioerythritol (DTE) decreased the electrophoretic mobility of Tsp, indicating reduction of an intrachain disulfide bond; Ca2+ prevented this effect. Electrophoresis of single-chain Tsp prepared with 50 mM DTE in either EDTA or Ca2+ also revealed a Ca2+-stabilized intrachain disulfide bond. Ca2+ prevented the retention of Tsp on an activated thiol-Sepharose column, indicating protection of a thiol by Ca2+. Incubation at 37 degrees C for 60 min resulted in complexes with apparent mass much greater than 500 kDa. Formation of complexes was prevented by N-ethylmaleimide, by a temperature less than 25 degrees C, and by Ca2+ or Mg2+. From pH 6 to 9, complexes formed better at lower pH. Two-dimensional (nonreduced/reduced) electrophoresis revealed Tsp but no other constituents of the complexes. With 10 nM thrombin, complexes formed faster and included thrombin; Ca2+ only partially inhibited. The complex was very susceptible to dissociation by low concentrations (2.5 mM) of DTE. It is concluded that Tsp has a reactive thiol and an intrachain disulfide bond that are protected by Ca2+. When these groups are unprotected, there is intermolecular thiol-disulfide exchange.

Blood Platelets↗
PubMed1985

Analysis of the fate of platelet-bound thrombin.

The thrombin-platelet interaction was investigated by analysis of the changes in the nature of platelet-associated thrombin during incubations for as long as 30 min. Washed human platelets were incubated with 125I-labeled human alpha-thrombin at either 22 or 37 degrees C. The saturably bound thrombin (total bound minus that bound in the presence of hirudin) was measured after collection of the platelets by centrifugation through a nonaqueous fluid. The rate of dissociation of bound thrombin was measured by addition of hirudin at intervals before centrifugation of the thrombin-platelet mixtures. Four states of thrombin-platelet complexes were identified: an initial rapidly equilibrating state; a more slowly dissociating state that formed within 5 min; and a nondissociable state and a large sodium dodecyl sulfate-stable complex that formed within 30 min. Transition from the rapidly equilibrating to the slowly dissociable state required a period of occupancy of activated platelets, but it did not require catalytically active thrombin. The nondissociable state represented 50-80% of the total saturably bound thrombin after a 30-min incubation at 37 degrees C. It formed only slightly at 22 degrees C or with inhibited thrombin. Formation of the sodium dodecyl sulfate-stable complex represented about 50% of platelet-associated thrombin after 30 min at 37 degrees C; only slight amounts were detected after incubation at 22 degrees C or with inhibited thrombin. The sodium dodecyl sulfate-stable complex was also found in the supernatant solution, with only about 20% of the total complex bound to platelets. Immunoprecipitation revealed that the complex included the platelet alpha-granule protein glycoprotein G (thrombin-sensitive protein, thrombospondin). It was concluded that only the initial rapidly equilibrating thrombin-platelet association could include binding necessary for platelet activation; transition to the other states requires platelet activation. By better describing the changes that occur in the nature of the binding of thrombin to platelets, this study explains most of the large discrepancies among published descriptions of the binding of thrombin to platelets.

Adenosine Diphosphate↗
PubMed1985

Differential effect of Serratia protease on platelet surface glycoproteins Ib and V.

The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease-pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.

Blood Platelets↗
PubMed1984

Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange.

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.

Blood Platelets↗
PubMed1984

Succinate potentiates the action of platelet agonists.

Succinate potentiated the actions of ADP, epinephrine, and the endoperoxide analog U46619 on platelets in plasma. Potentiation was observed at concentrations as low as 400 microM. Potentiation occurred immediately and did not change during incubations as long as five min. It was also observed with maleate and malonate but not with fumarate, oxalate, glutarate or succinate analogs with one or both carboxyls blocked. Succinate was able to reverse the inhibition of platelet activation due to elevated cAMP, and succinate and maleate were comparable to ADP in their ability to inhibit PGI2-induced synthesis of cAMP.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1984

Photoaffinity labeling of platelet thrombin-binding proteins.

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins: platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequence of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or alpha 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.

Blood Platelets↗
PubMed1984

Quantitative adsorption of platelet glycoprotein G (thrombin-sensitive protein, thrombospondin) to barium citrate.

Glycoprotein G (thrombin-sensitive protein, thrombospondin) is a high-Mr calcium-sensitive protein secreted by activated platelets. We observed that this protein was precipitated with barium citrate, and this property was used to purify glycoprotein G. The simple and rapid purification procedure consisted of barium citrate adsorption followed by heparin-agarose affinity chromatography. Unlike other calcium-sensitive proteins that are precipitated by barium citrate, glycoprotein G does not contain gamma-carboxyglutamic acid. The ability of glycoprotein G to bind to both heparin and barium citrate is consistent with this protein possessing clusters of positive and negative charges.

1-Carboxyglutamic Acid↗
PubMed1983

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation.

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.

Adenosine Triphosphate↗
PubMed1983

Characterization of the thrombin-induced desensitization of platelet activation by thrombin.

Brief exposure of platelets to thrombin makes them less sensitive to subsequent activation by thrombin, a phenomenon demonstrated by Shuman, Botney, and Fenton [J. Clin. Invest., 63, 1211-1218, 1979] by incubating prostacyclin-inhibited platelets with thrombin; after removal of thrombin and prostacyclin, the platelets were selectively desensitized to subsequent activation by thrombin. The conditions for this desensitization have been further defined. Inhibition of thrombin-induced platelet activation by prostacyclin was not absolute, it was only temporary, it could be overcome with higher thrombin concentrations, and it varied with platelet concentration and temperature. With low enough thrombin concentrations, high enough prostacyclin concentrations and short enough times of exposure, platelets could be pretreated with thrombin with no evidence of activation. After addition of hirudin to inhibit thrombin, the platelets were washed and tested for thrombin-induced secretion of ATP. Desensitization to thrombin depended on the concentration of thrombin during pretreatment and on the length of pretreatment, consistent with a catalytic modification of a receptor. A less extensive desensitization was observed when platelets without inhibitor were incubated with a sub-threshold level of thrombin before addition of an activating concentration of thrombin. This desensitization also varied with the time of pretreatment and the concentration of sub-threshold thrombin.

Blood Platelets↗
PubMed1983

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation.

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.

Binding Sites↗
PubMed1983

The action of calcium-dependent protease on platelet surface glycoproteins.

The action of exogenous calcium-dependent protease (CDP) on tritium-labeled surface glycoproteins was analyzed by incubation of labeled, washed human platelets with CDP partially purified from human platelets. Labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Incubation of the labeled platelets with the protease led to a loss (calcium-dependent) from the platelets of glycoproteins Ib and V and concomitant appearance in the supernatant solution of glycocalicin (a proteolytic fragment of glycoprotein Ib), glycoprotein V, and other, unidentified glycoproteins. These changes in surface label were accompanied by alterations in three parameters of platelet function. Compared to control platelets, the CDP-treated platelets were activated by thrombin more slowly and showed less saturable and nonsaturable binding of thrombin. The CDP-treated platelets, but not the controls, aggregated on addition of fibrinogen, indicating that treatment with CDP had exposed fibrinogen receptors. The alterations in surface glycoproteins and functional parameters were compared over a 1000-fold range of CDP treatment. The decreased binding of thrombin and the exposure of fibrinogen receptors were correlated with the release of surface glycoproteins to the supernatant solution, but the slow activation by thrombin was observed under conditions where no release of labeled glycoproteins was detected (i.e., brief incubations with low concentrations of CDP). Activation of the endogenous CDP with 2.5 mM calcium chloride plus the ionophore A23187 was accompanied by hydrolysis of actin-binding protein, a known substrate, and release to the supernatant solution of labeled glycocalicin and glycoprotein V plus a faster-migrating glycoprotein not released by exogenous protease. This effect was observed in the presence of leupeptin, which completely inhibited action of exogenous protease, suggesting that platelet calcium-dependent protease may modify the platelet surface in ways that can cause alterations of platelet function.

Adenosine Triphosphate↗
PubMed1981

Characteristics of the synergistic actions of platelet agonists.

Platelets are activated by many different agonists that act synergistically. Since there is a characteristic pattern of responses to each agonist, and since there is a clear distinction between weak and strong agonists, understanding the nature of the synergism and its physiologic significance requires characterization of the pattern of responses to the synergistic action of the various agonists. Shape change, aggregation, and secretion of ATP by human platelets in citrated plasma were analyzed after activation by ADP, epinephrine, arachidonic acid, gamma-thrombin, or collagen, either singly or in pairs. The patterns of responses were characteristic of the agonist in higher concentration relative to its threshold concentration; if neither was clearly higher, the pattern of responses was intermediate between the responses characteristic of each agonist. No combination of weak agonists had the characteristics of a strong agonist. These results help define the extent to which platelet responses can be attributed to the synergistic actions of weak agonists.

Adenosine Diphosphate↗
PubMed1980

Reassessment of the evidence for the role of secreted ADP in biphasic platelet aggregation. Mechanism of inhibition by creatine phosphate plus creatine phosphokinase.

The observation that platelet secretion occurs in parallel with the second wave of platelet aggregation necessitates reassessment of the generally accepted concept that secreted ADP is the cause of second-wave aggregation. The major evidence for an involvement of secreted ADP is inhibition of second-wave aggregation by enzyme-catalyzed removal of ADP, interpreted as removal of secreted ADP. An alternative hypotheses, that the observed inhibition is due to a decrease in potentiation by extracellular ADP present prior to addition of stimulus, has been tested with the enzyme system CP plus CPK (CP/CPK). Low levels of CP/CPK inhibited gamma-thrombin-induced second-wave aggregation only after preincubation. When the second wave of gamma-thrombin- or epinephrine-induced aggregation was inhibited by very high levels of CP/CPK, the inhibition was overcome by an increase in the level of stimulus. These results are inconsistent with the idea that CP/CPK blocks second-wave aggregation by removing secreted ADP, but they are consistent with a decreased potentiation of the platelets due to a lower level of extracellular ADP prior to stimulation. Freshly prepared platelet-rich plasma contained a mean (n = 16) of 12 nM extracellular ADP. These data demonstrate that inhibition by CP/CPK cannot be taken as evidence for the involvement of secreted ADP.

Adenosine Diphosphate↗
PubMed1979

Dissociation of thrombin from platelets by hirudin. Evidence for receptor processing.

Hirudin inhibited the binding of human 125I-alpha-thrombin to the saturable, but not the nonsaturable, sites on washed human platelets. When hirudin was added to a thrombin-platelet mixture, it caused a biphasic dissociation of bound thrombin. A partial dissociation was too rapid to measure and was followed by complete dissociation with a first order rate constant of about 10(-2) s-1. The fraction of bound thrombin in the more slowly dissociable form increased from essentially none after a 5-s preincubation of thrombin and platelets to as much as 75% of saturable binding after a 4-min preincubation. Transition to the slowly dissociable state was not accompanied by an increase in the amount bound and was not observed with active site serine-derivatized thrombin. This is the first evidence with intact platelets of a binding characteristic that depends, as does platelet stimulation, on catalytically active thrombin, suggesting that it may represent physiologically significant receptor processing.

Blood Platelets↗