Increased phosphorylation of human fibrinopeptide A under acute phase conditions

The High Performance Liquid Chromatography (HPLC) separation of the fibrinopeptides liberated by the action of thrombin from plasma fibrinogen in a new one-step procedure without prior purification of fibrinogen is described. Since the phosphorylated and non-phosphorylated form of fibrinopeptide A are clearly resolved by this method, the determination of the degree of phosphorylation of fibrinopeptide A from the peak heights of these peptides becomes possible. By this method the degree of phosphorylation of fibrinogen in healthy volunteers (n = 21) is found to be 23.6 +/- 3.6%. Under acute phase conditions where the synthesis rate of fibrinogen is known to be markedly enhanced the degree of its phosphorylation also increases considerably. This was demonstrated on 13 patients undergoing an elective hip joint replacement, the hip surgery being chosen as a model for the elicitation of an acute phase reaction. The degree of phosphorylation rises steeply up to 60% on the first day after operation thereafter declining slowly to normal values within about one week. The maximum of the degree of phosphorylation precedes that of the fibrinogen concentration by several days. The mechanism which leads to the higher phosphorylation of fibrinogen during increased synthesis is unknown at the moment.     

The location of a second in vivo phosphorylation site in the A alpha-chain of human fibrinogen

By quantitative phosphorus determination on the single chains of human fibrinogen it is demonstrated that the covalently bound phosphorus of adult and fetal fibrinogen is exclusively located in the A alpha chain. The A alpha-chain of fetal fibrinogen contains about twice as much phosphorus as the adult A alpha-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the A alpha-chain. By consecutive cleavage of the A alpha-chains of fetal and adult fibrinogen with cyanogen bromide, trypsin, and chymotrypsin, separation of the resulting peptide mixtures and analysis for phosphorylated amino acids, this second phosphorylation site could be traced to Ser 345 of the A alpha-chain. There is only one sequence homology between the two now known in vivo phosphorylation sites of human fibrinogen, namely that the second amino acid to the carboxyl side of the phosphorylated Ser is Glu. The sequence specificity of the up to now unidentified protein kinase phosphorylating fibrinogen allows it to be classified as a member of the group of type-2 casein kinases or casein kinases TS.     

Novel axonal distribution of neurofilament‐H phosphorylated at the glycogen synthase kinase 3β‐phosphorylation site in its E‐segment

The Ser493 residue in the E‐segment of the rat neurofilament heavy chain (NF‐H) is phosphorylated by glycogen synthase kinase 3β (GSK3β) in vitro and in spinal cord. We examined Ser493 phosphorylation by analyzing developmental changes and cellular distribution of phospho‐Ser493 using phosphorylation‐site‐specific antibodies. This residue was phosphorylated in NF‐H prepared from human, rat, and mouse spinal cord, all species in which the amino acid sequence of NF‐H is known. Phosphorylated Ser493 appeared on postnatal day 2 in rat brain, at the same time when NF‐H is first detected. It gradually increased together with the increase in total NF‐H during brain development. Phospho‐Ser493 was detected on the phosphorylated form of NF‐H at multiple Lys‐Ser‐Pro (KSP) repeats, which are distributed mainly in axons. In rat ventral horn, phosphorylated Ser493 was localized in axons but not in cell bodies or dendrites. However, the distributions of phosphorylated Ser493 and KSP phosphorylation in axons were not identical. Ser493 was continuously phosphorylated at nodes of Ranvier, whereas the KSP sites were dephosphorylated. Ser493 was also phosphorylated in unmyelinated regions of optic nerve axons. A biochemical difference in phosphorylation between Ser493 and KSP repeats was also found; the subtle phosphorylation at Ser493 was detected in NF‐H unphosphorylated at the KSP repeats by immunoblotting cerebral cortex extracts. These results indicate that Ser493 in the NF‐H E‐segment is a novel site that is phosphorylated in both the myelinated and the unmyelinated regions of axons. © 2009 Wiley‐Liss, Inc.     

The enhanced activation of Glu-plasminogen by urokinase in the presence of fibrin or des A fibrin as measured by the release of B beta peptide and FDP

Fresh plasma was incubated either with urokinase (UK) alone or the mixture of UK and human thrombin or Reptilase. In a purified system Glu-plasminogen (Glu-plg) was incubated with fibrinogen or fibrinogen plus thrombin in the presence of UK. At intervals, aprotinin was added to stop the reactions and the amounts of B beta peptide and fibrin(ogen) degradation products (FDP, FgDP) were measured by radioimmunoassay and enzyme immunoassay, respectively. Results obtained by using plasma showed that fibrin was degraded faster than fibrinogen upon the addition of UK to the plasma or plasma clotted with thrombin. B beta peptide was released faster from the clot than plasma. The clot formation caused by Reptilase (release of fibrinopeptide A) was accompanied by increase in the release of B beta 1-42 from des A fibrin (fibrin without fibrinopeptide A). In a purified system, fibrin was degraded faster than fibrinogen upon the activation of Glu-plg by UK. These results may correlate well with the observation that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen.     

The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen

The alpha-chain of human fibrinogen was found to be phosphorylated in EDTA-anticoagulated whole blood when trace amounts of (gamma-32P)ATP and 7.5 mM Mg2+ ions were added. Fibrinogen was not phosphorylated if only the ATP was added. The thrombin-induced gelation of fibrinogen phosphorylated by protein kinase A, casein kinase I or II was studied spectrophotomerically. It was found that phosphorylation by protein kinase A caused the formation of thinner fibrin fibres, whereas phosphorylation by casein kinase II resulted in fibres slightly thicker than those of the control fibrinogen (equivalent to a 20% increase in the control fibrinogen concentration). Phosphorylation with casein kinase I did not significantly affect the fibrin fibre thickness. Dephosphorylation by alkaline phosphatase removed 50% of the 32P-labelled phosphate from protein kinase A-phosphorylated fibrinogen and over 90% from the casein kinase I or II-phosphorylated fibrinogens. This dephosphorylation resulted in a general increase in fibre thickness in the gelation assay in all samples, although the fibres of the phosphorylated fibrinogens remained substantially thinner than the dephosphorylated control fibrinogen. Plasmin digestion of the phosphorylated fibrinogens showed that they were more resistant to cleavage, being cleaved at only 30% to 70% of the rate of control fibrinogen and that this resistance was unaltered by dephosphorylation, in contrast to the thrombin gelation experiments.     

Plasmin digestion of human fibrinogen previously phosphorylated by protein kinase C or dephosphorylated by alkaline phosphatase in vitro

Human fibrinogen, either untreated or previously phosphorylated by protein kinase C, was incubated with plasmin generated by streptokinase, urokinase or tissue plasminogen activator and the resulting fragments were separated by gel electrophoresis. Plasmin degradation resulted in the expected X, Y and D fragments, but the degradation rates differed. In vitro phosphorylation of fibrinogen was seen to inhibit the plasmin digestion. Treatment with alkaline phosphatase did not reverse the inhibition.     

Synthesis and radioimmunoassay of canine fibrinopeptide A

Canine fibrinopeptide A, the 16 residue NH₂-terminal segment of the Aα chain of canine fibrinogen released by thrombin proteolysis and its NH₂-tyrosyl analogue, were synthesized by stepwise solid-phase syntheses. Following cleavage, the synthetic peptides were purified by ion-exchange chromatography, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis and Edman degradation analysis. Rabbits immunized with carbodiimide-coupled synthetic canine fibrinopeptide A-bovine albumin conjugates produced antibodies which bound up to 70% of available counts using ¹²⁵I-N-tyrosyl canine fibrinopeptide A as tracer. Quantitative displacement of bound tracer could be effected by both synthetic canine fibrinopeptide A and clot supernates prepared from purified canine fibrinogen. Fifty percent displacement of radiolabelled tracer could be achieved with 1.1 picomole of peptide antigen. Antiserum to canine fibrinopeptide A was about 50-fold less sensitive on a molar basis to canine fibrinogen as compared to the free peptide in solution. This suggests that antigenic determinants on the canine fibrinopeptide are altered by attachment of the peptide to its parent molecule. The molar reactivity of human fibrinopeptide A was about 25% in comparison with canine fibrinopeptide A, indicating that both the human and canine peptides share some antigenic determinants. Using this assay, fibrinopeptide A immunoreactivity was measured in canine plasma following removal of fibrinogen by ethanol precipitation and dialysis. The mean fibrinopeptide A level 4 normal dogs (18 determinations) was found to be 1.5±0.98 pmols/ml anticoagulated plasma.     

pRb phosphorylation is regulated differentially by cyclin-dependent kinase (Cdk) 2 and Cdk4 in retinoic acid-induced neuronal differentiation of P19 cells

The retinoblastoma protein (pRb) is a key regulator of cell growth, differentiation and survival. pRb(-/-) mice show abnormal neuronal cell death in the developing brain. The function of pRb is regulated by its phosphorylation state. In this study, the phosphorylation of pRb during retinoic acid (RA)-induced neuronal differentiation of P19 cells was examined using site-specific antibodies against pRb phosphorylated at Ser601, Ser605 and Ser773. Although pRb was hyperphosphorylated in undifferentiated P19 cells, Ser601 and Ser773 were not phosphorylated. Upon exposure to RA, however, these two sites became strongly phosphorylated. Cdk4 kinase activity was almost undetectable in undifferentiated P19 cells, but was strongly activated on exposure to RA. In contrast, Cdk2 kinase activity and the phosphorylation of Ser605 were observed in undifferentiated cells as well as in RA-treated cells. These observations suggest that Cdk2 and Cdk4 may phosphorylate different sites of pRb in vivo and that the two sites of pRb examined here are newly phosphorylated during RA-induced neuronal differentiation in P19 cells.     

Fibrinogen Zurich I: impaired release of fibrinopeptide A.

Fibrinogen Zurich I is characterized by an abnormal fibrin monomer polymerization. It consists of two fractions of molecules, one with a normal aggregation and one not aggregating at all and interfering with the aggregation of the normal population. Using a radioimmunoassay for fibrinopeptide A, only approximately half of the expected fibrinopeptide A could be recovered after thrombin or Defibrase proteolysis. The defective fibrinopeptide A release could be confirmed by measurement of the N-terminal Gly/Tyr ratio. It is likely that the abnormal fibrin monomer aggregation of the abnormal fraction of fibrinogen Zurich I is due to the defective fibrinopeptide A release of this fraction.     

Protection by cilostazol against amyloid-β(1-40)-induced suppression of viability and neurite elongation through activation of CK2α in HT22 mouse hippocampal cells

Amyloid-β peptide (Aβ) deposits in the brain are critical in the neurotoxicity induced by Aβ. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aβ(1-40) (3-30 μM)-induced deterioration of cell proliferation, viability, and neurite elongation. Cilostazol rescued HT22 cells from the apoptotic cell death induced by Aβ toxicity through the downregulation of phosphorylated p53 (Ser15), Bax, and caspase-3 and the upregulation of Bcl-2 expression, which improved neuronal cell proliferation and viability. Furthermore, Aβ(1-40) suppressed both phosphorylated CK2α protein expression and CK2 activity in the cytosol; these were concentration dependently recovered by cilostazol (3-30 μM). Cilostazol significantly increased the levels of GSK-3β phosphorylation at Ser9 and β-catenin phosphorylation at Ser675 in the cytosol and nucleus. Cilostazol effects were reversed by KT5720 (1 μM, PKA inhibitor) and TBCA (40 μM, inhibitor of CK2) and CK2α knockdown by siRNA transfection. Likewise, Aβ-stimulated GSK-3β phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aβ (10 μM)-induced suppression of neurite elongation was recovered by cilostazol; this recovery was attenuated by inhibitors such as KT5720 and TBCA and blocked by CK2α knockdown. In conclusion, increased cAMP-dependent protein kinase-linked CK2α activation underlies the pharmacological effects of cilostazol in downregulating p53 phosphorylation at Ser15 and upregulating GSK-3β phosphorylation at Ser9/β-catenin phosphorylation at Ser675, thereby suppressing Aβ(1-40)-induced neurotoxicity and improving neurite elongation.     

Fibrinogen petoskey: Identification of a new dysfibrinogenemia characterized by altered release of fibrinopeptide A

A new congenital dysfibrinogenemia, designated fibrinogen Petoskey, which was traced through four generations of a Michigan family, was found to exhibit an abnormally slow rate of release of fibrino-peptide A upon treatment with thrombin and batroxobin. Batroxobin only partially hydrolyzed and polymerized the fibrinogen from affected individuals, suggesting that these patients had both normal and abnormal fibrinogen in their circulation and that batroxobin was not capable of releasing fibrinopeptide A from the abnormal fibrinogen. Polymerization of fibrin monomers from fibrinogen Petoskey and plasmin mediated digestion of fibrinogen Petoskey were normal. The Factor XIIIa-catalyzed cross-linking of fibrinogen Petoskey was slightly delayed at low (but not at high) concentrations of thrombin. This delayed cross-linking appeared to be a secondary effect of the lower rate of release of fibrinopeptide A.     

The interrelationship between selective tau phosphorylation and microtubule association

The purpose of this study was to examine the modulation of tau phosphorylation mediated by protein kinase A, a kinase with low intrinsic activity, and by the constitutively active glycogen synthase kinase, as well as to examine the subsequent effects on tau-microtubule association in differentiated human SH-SY5Y neuroblastoma cells. Activation of protein kinase A with forskolin and rolipram significantly increased tau phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rapidly. In contrast, glycogen synthase kinase appears to maintain tau phosphorylation at Thr181 and Ser396/404 since inhibition of glycogen synthase kinase with lithium reduced phosphorylation at these sites. Lithium treatment also significantly decreased tau and tyrosinated α-tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, indicating that both constitutive kinase activity and microtubule state modulate tau phosphorylation at these sites. Nocodazole- or taxol-induced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibitor cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epitope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 extraction assay was utilized to separate the detergent-soluble cytosolic components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detected in the cytosolic fraction. Activation of protein kinase A in the presence of okadaic acid elevated tau levels in the detergent-soluble fraction, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetylated α-tubulin levels. In conclusion, the phosphorylation state of tau in differentiated SH-SY5Y cells is regulated by glycogen synthase kinase, microtubule dynamics and osmotic stress at overlapping sites which apparently have little influence on tau-microtubule association. In contrast, phosphorylation of tau at Ser262/356 within the microtubule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ.     

The effect of temperature on formation of fibrin complexes

The stability of soluble fibrin complexes to dissociation by temperature increase is dependent on the structure of the complexes. Plasmin degradation of fibrin clots leads to the formation of soluble complexes, the stability of which depend on their content of fibrinopeptide B. Complexes lacking only fibrinopeptide A dissociate readily at 37°C whereas those lacking fibrinopeptides A and Bare stable at 37°C. Removal of the carboxyterminal part of the A () chain of fibrinogen by plasmin results in decreased stability of soluble complexes formed by those degraded fibrinogen molecules.     

Microtubule-affinity regulating kinase (MARK) is tightly associated with neurofibrillary tangles in Alzheimer brain: a fluorescence resonance energy transfer study

Paired helical filaments, the main structural components of the neurofibrillary tangles in Alzheimer disease, consist of phosphorylated tau protein. Because the levels and degree of phosphorylation are significantly higher in paired helical filament (PHF)-derived tau than in normal adult tau, and because phosphorylation of tau severely disrupts microtubule stability, it is postulated that tau phosphorylation is an important step in PHF formation. The kinases and/or phosphatases that act in vivo to help induce such a pathological state of tau, however, are not yet known. In this study we implicate the non-proline directed kinase MARK in PHF-tau phosphorylation, by virtue of its close intermolecular association with the phosphorylated Ser262 epitope on PHF-tau as assessed by fluorescence resonance energy transfer. Moreover, because this tight enzyme-substrate association is observed in neurofibrillary tangles in Alzheimer tissue, we suggest that PHF-tau phosphorylation may occur to some extent on assembled PHF filaments.     

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.     

Fibrinogen Kawaguchi: an abnormal fibrinogen characterized by defective release of fibrinopeptide A

A congenital dysfibrinogenemia was found in a 32-year-old asymptomatic female and her immediate family. The propositus, apparently a heterozygote for the abnormality, characteristically showed defective release of fibrinopeptide A from half of her fibrinogen molecules. No fibrinopeptide A was cleaved off from the isolated abnormal molecule by thrombin or snake venoms (Reptilase and Ancrod) as evidenced by radioimmunoassay, high performance liquid chromatography and determination of the NH2-terminal amino acids. The abnormal fibrinogen formed a solid gel solely by the release of fibrinopeptide B upon incubation with thrombin. We provisionally designate this abnormal fibrinogen as "Fibrinogen Kawaguchi", although possible identity with other abnormal fibrinogens is not excluded.     

Fibrinogen Bergamo I (A alpha 16Arg----Cys): susceptibility towards thrombin following aminoethylation, methylation or carboxamidomethylation of cysteine residues

An abnormal fibrinogen, denoted as "fibrinogen Bergamo I", has been characterized. Its defect consists in an exchange of arginine by cysteine in position 16 of the A alpha-chain, thus corresponding to that found in a number of other fibrinogen variants. The abnormal fibrinopeptide A cannot be split off by thrombin from intact fibrinogen Bergamo I. We describe three different chemical modifications of the cysteine A alpha 16, i.e. aminoethylation, methylation and carboxamidomethylation, and their effects on the susceptibility of fibrinogen Bergamo I towards thrombin attack. S-aminoethylation of the A alpha 16Cys renders the peptide bond A alpha 16-17 cleavable by thrombin. Following methylation or carboxamidomethylation, the A alpha 19-arginyl bond becomes accessible for thrombin. The chemically modified extended fibrinopeptide A can be readily separated from the normal fibrinopeptide A by HPLC. The latter two modifications are suitable alternative procedures for detecting the molecular defect A alpha 16Arg----Cys of fibrinogen.     

Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein

alpha-Synuclein (alpha-syn) is the major component of pathologic inclusions that characterize neurodegenerative disorders such as Parkinson disease, dementia with Lewy body disease, and multiple system atrophy. The present study uses novel phospho-specific antibodies to assess the presence and regulation of phosphorylated Ser87 and Ser129 in alpha-syn in human brain samples and in a transgenic mouse model of alpha-synucleinopathies. By immunohistochemistry, alpha-syn phosphorylated at Ser129, but not at Ser87, was abundant in alpha-syn inclusions. Under normal conditions, Ser129 phosphorylation, but not Ser87 phosphorylation, was detected at low levels in the soluble biochemical fractions in human alpha-syn transgenic mice and stably transfected cultured cells. Therefore, a role for Ser87 phosphorylation in alpha-synucleinopathies is unlikely, and in vitro assays showed that phosphorylation at this site would inhibit polymerization. In vitro studies also indicated that hyperphosphorylation of Ser129 alpha-syn in pathologic inclusions may be due in part to the intrinsic properties of aggregated alpha-syn to act as substrates for kinases but not phosphatases. Further studies in transgenic mice and cultured cells suggest that cellular toxicity, including proteasomal dysfunction, increases casein kinase 2 activity, which results in elevated Ser129 alpha-syn phosphorylation. These data provide novel explanations for the presence of hyperphosphorylated Ser129 alpha-syn in pathologic inclusions.     

Radioimmunoassay of fibrinopeptide A-clinical applications.

The radioimmunoassay technique developed by Nossel et al. for measuring fibrinopeptide A released by thrombin action on fibrinogen was used for assessing the degree of intravascular coagulation in patients. Plasma fibrinopeptide A values in 36 normal persons were below 2 ng/ml. Elevated levels were often found in cases of malignancies, burns and fractures, whereas uremic patients usually had normal values. Samples could be stored for several weeks without significant loss of immunoreactivity.     

Lithium inhibits stress-induced changes in tau phosphorylation in the mouse hippocampus

Summary. It has been pointed out that hyperphosphorylation of microtubule-associated protein tau caused by stress might participate in the early stages of Alzheimer’s disease pathogenesis. In the present study, we investigated the effects of cold water stress (CWS) on tau phosphorylation in the mouse hippocampus and the effects of GSK-3β inhibitor, LiCl, on CWS-induced changes in tau phosphorylation levels by immunoblot analyses. CWS exposure caused an increase in tau phosphorylation at the Tau-1 (Ser199/202), AT8 (Ser202/Thr205) and Ser396 sites. CWS-induced changes in tau phosphorylation at the Ser199/202 and Ser396, but not at Ser202/Thr205, were significantly attenuated by LiCl pretreatment. Total tau levels also showed a decided tendency to increase after CWS, which tendency was also countered by LiCl. Finally, we showed that CWS increased the active form of GSK-3β that was phosphorylated at Tyr216. These results suggest that a CWS-induced increase in phosphorylated tau in the hippocampus is mediated, at least partly, by the activation of GSK-3β.