Measurement of soluble transferrin receptor in serum of healthy adults.

The concentration of soluble transferrin receptor (sTfR) in serum is reported to be useful in the diagnosis of iron deficiency, especially for patients with concurrent chronic disease, where routine tests of iron status are compromised by the inflammatory condition. A new diagnostic assay for sTfR is calibrated against natural plasma sTfR, thus minimizing calibration discrepancies that result from differences between the analyte and the cellular transferrin receptor used in other assays. Use of the new assay to measure sTfR concentrations in 225 healthy, hematologically normal adults provided a reference interval against which pathological samples could be compared. There was no difference in the reference intervals for men and women and no correlation of [sTfR] with the age of the subject. Black subjects had significantly higher concentrations than nonblacks, and people living at high altitude had higher concentrations than those living closer to sea level. These differences were additive.

Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase.

Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [3H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys974 was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37 degrees C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.

Characterization of protein disulphide isomerase released from activated platelets.

Protein disulphide isomerase (PDI) activity is released by activated platelets. In this study, PDI was purified from platelets and found to have an apparent mass, pI and N-terminal sequence similar to those for other human PDIs. Rabbit antibodies were generated and used to establish that, on activation, platelets release a protein immunologically identical to PDI in platelets. Approximately 10% of total platelet PDI was released by thrombin and 20% by calcium ionophore. The antibody was used to demonstrate PDI on the external surface of platelets by electron microscopy. Flow cytometry was used to demonstrate that upon activation of platelets with ionophore PDI was released by vesiculation. Since platelets are present and become activated at sites of vascular injury, platelet PDI may play a role in the various haemostatic and tissue remodelling processes in which platelets are involved.

Reactions of thrombin-serpin complexes with thrombospondin.

Activated platelets release proteins that form stable complexes with thrombin (J. J. Miller, P. C. Browne, and T. C. Detwiler, Biochem. Biophys. Res. Commun. 151, 9-15, 1988). A working model for the reaction (P. C. Browne, J. J. Miller, and T. C. Detwiler, Arch. Biochem. Biophys. 265, 534-538, 1988) includes a dissociable complex of thrombin with released platelet protease nexin, leading to formation of a nondissociable thrombin-nexin complex that then becomes disulfide linked to thrombospondin. This disulfide-linked complex is converted back to the thrombin-nexin complex by reduction of disulfide bonds. Results that allow elaboration on this model are presented. After longer periods of incubation or after incubation with higher concentrations of thrombin, the amount of thrombin complexed with thrombospondin exceeded the amount of thrombin-nexin complex recovered after reduction of disulfide bonds. When the reaction mixture included inhibitors of formation of the thrombin-nexin complex, a slow formation of the thrombin-thrombospondin complex was observed. It was concluded that there is a nexin-independent as well as the faster nexin-dependent disulfide linkage of thrombin to thrombospondin. Addition of thrombin-antithrombin III complexes to the supernatant solution of activated platelets also led to complexes with thrombospondin, demonstrating that serpins other than platelet protease nexin facilitate incorporation of thrombin into complexes with thrombospondin. By heparin affinity chromatography, it was shown that thrombin-nexin complexes dissociably associate with thrombospondin prior to formation of disulfide-linked complexes. These observations are incorporated into a more detailed model of the reaction.

Protein disulfide isomerase activity is released by activated platelets.

The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin-thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

Ester and amide derivatives of E64c as inhibitors of platelet calpains.

Ester and amide derivatives of E64c, (+)-(2S,3S)-3-[[(S)-3-methyl-1- [(3-methylbutyl)carbamoyl]butyl]carbamoyl]-2-oxiranecarboxylic acid, an inhibitor of calpains, were synthesized and tested for ability to inhibit calpain in lysed cells, ability to enter intact cells, and ability to inhibit calpain in intact cells. The esters were from halogen-substituted alcohols and alcohols with increasing size. There were no appreciable differences in the inhibitory potency of any of the halogen-substituted esters from ethyl to trifluoroethyl, indicating that ease of hydrolysis of this class of ester is not important for activity. The only ester with impaired activity was the largest, Z-leucyl-norleucyl, which was about 5% as effective as the ethyl ester, E64d. Amides of amino acid esters also had impaired activity. To explore the possibility of targeting E64c derivatives to specific cells, esters and amides of E64c with 5-hydroxytryptamine were tested on the rationale that the active 5-hydroxytryptamine uptake mechanism of platelets might selectively concentrate the drug in platelets. Both the ester and amide inhibited calpain in lysed cells, but only the ester inhibited in intact cells. The 5-hydroxytryptamine ester showed no advantage over the ethyl ester in entering platelets.

The reaction of thrombin with platelet-derived nexin requires a secondary recognition site in addition to the catalytic site.

A protease nexin released by activated platelets forms stable complexes with alpha-thrombin. Active-site-blocked thrombin does not form the stable complex, but it inhibits formation of the stable complex by active alpha-thrombin. gamma-Thrombin, which has a damaged substrate recognition site (the anion-binding exosite), did not form the complex and did not inhibit formation of the stable complex by alpha-thrombin. Complex formation was inhibited by the C-terminal dodecapeptide of hirudin, which has been shown to bind to the anion-binding exosite. A monoclonal antibody that blocks reactions of thrombin that involve the anion-binding exosite also inhibited formation of a stable complex of alpha-thrombin and the platelet-derived protease nexin. It is concluded that the anion-binding exosite of thrombin, a site that confers a high degree of specificity for substrates with a complementary site, binds to the platelet nexin prior to reaction of the catalytic site with the serpin.

Reduction of a disulfide bond of thrombospondin in the supernatant solution of activated platelets.

Incubation of the material secreted by activated platelets leads to the formation of disulfide-linked dimers and multimers of one of the proteins, thrombospondin. To determine whether these complexes formed as a result of thiol-disulfide exchange (no change in the number of thiols) or of oxidation of thiols (a decrease in the number of thiols), the number of thiols in TSP was measured during formation of multimers. The number of thiols increased from about 3/mol to 4.8/mol. The half-time for the disappearance of monomers of thrombospondin was fourfold greater than the half-time for appearance of new thiols. The appearance of new thiols, as well as the formation of multimers, was inhibited by Ca2+. The appearance of new thiols was reversible; addition of Ca2+ reversed the process, and at pH 8, but not at pH 6 or 7, the appearance of new thiols spontaneously reversed. No new thiols formed during incubation of partially purified thrombospondin or after the supernatant solution had been treated with activated thiol-Sepharose to remove reactive thiol compounds. It is concluded that thrombospondin has a disulfide bond that is unstable in the absence of Ca2+. It can be attacked by a thiol of another molecule of thrombospondin to form disulfide-linked multimers, by a thiol of the same molecule of thrombospondin to generate isomerization of disulfide bonds or, as observed in this study, by another secreted thiol compound to give a mixed disulfide and a new thiol.

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization.

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.

Characteristics of a thrombin inhibitor secreted by activated platelets.

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.

Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d.

E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.

Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate.

Thrombin forms sodium dodecyl sulfate stable complexes of 77 and greater than 450 kDa with proteins secreted by activated platelets. The kinetics of formation of these complexes were investigated by addition of 125I-thrombin to the supernatant solution of A23187-activated platelets. Complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis either with or without reduction of disulfide bonds. When analyzed on nonreduced gels, the 77-kDa complex reached a maximum at about 3 min and then declined as the greater than 450-kDa complex increased. On reduced gels (on which there was no greater than 450-kDa complex) the 77-kDa complex approached the level of the greater than 450-kDa complex on nonreduced gels. The half-time of formation was less than 1 min for the 77-kDa complex and about 15 min for the greater than 450-kDa complex. These time courses suggested that the 77-kDa complex was incorporated into the greater than 450-kDa complex as an essential precursor. Formation of complexes was inhibited by a competitive inhibitor or a noncompetitive inhibitor of thrombin, and the pH dependence of formation of both complexes was similar to the pH dependence for catalytic activity of thrombin. Ca2+ inhibited formation of the greater than 450-kDa complex but not of the 77-kDa complex. A model is presented in which thrombin and a secreted protein form a 77-kDa complex by a process that involves the active site of thrombin. The 77-kDa complex is then incorporated into a greater than 450-kDa complex by thiol-disulfide exchange with thrombospondin, a process that is inhibited by Ca2+. Thrombin in the greater than 450-kDa complex had no catalytic activity.

Complexes of thrombin with secreted platelet proteins.

A labeled 77-kDa complex formed when 125I-thrombin was added to platelet suspensions or to the supernatant solution of ionophore-activated platelets. Prostacyclin inhibited complex formation with whole platelets but not with the supernatant solution of ionophore-activated platelets. This is evidence that the complex formed with a factor secreted from activated platelets. Smaller complexes of 70 and 58 kDa formed between labeled thrombin and lysed platelets. The 77-kDa complex was necessary for the formation of a thrombin-thrombospondin complex.

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.

The effects of lithium on platelet phosphoinositide metabolism.

The effects on phosphoinositide metabolism of preincubation of platelets for 90 min with 10 mM-Li+ were studied. Measurements were made of [32P]phosphate-labelled phosphoinositides and of [3H]inositol-labelled inositol mono-, bis- and tris-phosphate (InsP, InsP2 and InsP3). Li+ had no effect on the basal radioactivity in the phosphoinositides or in InsP2 or InsP3, but it caused a 1.8-fold increase in the basal radioactivity in InsP. Li+ caused a 4-, 3- and 2-fold enhanced thrombin-induced accumulation of label in InsP, InsP2 and InsP3 respectively. Although the elevated labelling of InsP2 and InsP3 returned to near-basal values within 30-60 min, the high labelling of InsP did not decline over a period of 60 min after addition of thrombin to Li+-treated platelets, consistent with inhibition of InsP phosphatase by Li+. The effect of Li+ was not due to a shift in the thrombin dose-response relationship; increasing concentrations of thrombin enhanced the initial rate of production of radiolabelled inositol phosphates, whereas Li+ affected either a secondary production or the rate of their removal. The only observed effect of Li+ on phosphoinositide metabolism was a thrombin-induced decrease (P less than 0.05) in labelled phosphatidylinositol 4-phosphate in Li+-treated platelets; this suggests an effect on phospholipase C. Li+ enhanced (P less than 0.05) the thrombin-induced increase in labelled lysophosphatidylinositol, suggesting an effect on phospholipase A2. It is concluded that Li+ inhibits InsP phosphatase and has other effects on phosphoinositide metabolism in activated platelets. The observed effects occur too slowly to be the mechanism by which Li+ potentiates agonist-induced platelet activation.

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.

Modified platelet responses to thrombin. Evidence for two types of receptors or coupling mechanisms.

The normally quick response of platelets to alpha-thrombin is delayed under two conditions. After pretreatment of platelets with chymotrypsin or certain other proteases, aggregation and secretion induced by alpha-thrombin begins after a delay of 30 s or more compared to less than 5 s for control platelets, and control platelets are activated by gamma-thrombin with a similar delay (Tam, S. W., Fenton, J. W., II, and Detwiler, T. C. (1980) J. Biol. Chem. 255, 6626-6632). Under these conditions, thrombin-induced inhibition of adenylate cyclase was blocked. The impaired regulation of adenylate cyclase and delayed secretion had in common: (i) partial correction with high concentrations of thrombin; (ii) similar thrombin dose-response relationships; (iii) similar concentration dependence for chymotrypsin during pretreatment; and (iv) specificity for thrombin. Other parameters of thrombin-induced platelet activation were also analyzed under these conditions. Thrombin-induced hydrolysis of arachidonyl esters and synthesis of prostanoids were similar to regulation of adenylate cyclase; they were blocked, with partial correction at high concentrations of thrombin. In contrast, thrombin-induced synthesis of phosphatidic acid and phosphorylation of a 20- and a 40-kDa protein were similar to secretion; they occurred after a delay but to a normal extent. The thrombin-induced increase in cytosolic calcium ion activity was slightly slower in chymotrypsin-treated platelets or in response to gamma-thrombin, but it was complete prior to initiation of the delayed responses. It is concluded that platelets have at least two types of thrombin receptors or coupling mechanisms, one of which is sensitive to chymotrypsin, unresponsive to gamma-thrombin, and coupled to inhibition of adenylate cyclase and activation of prostanoid synthesis.

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.