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Biomedical subjects

H Takeya

Publications and source records attributed to H Takeya.

At least 37 records · Page 2Linked to original sources
PubMed1996

Protein C activation in NIDDM patients.

Enhanced activation of the clotting system has been recently implicated in the pathogenesis of vascular complications in patients with diabetes mellitus. Abnormalities of the anticoagulant system may constitute a potential trigger factor for the haemostatic activation observed in diabetic subjects. The current study aimed to evaluate anticoagulant activity in diabetic patients by assessing the plasma levels of activated protein C-protein C inhibitor complex; and by measuring the anticoagulant response to exogenous thrombomodulin. This study comprised 61 patients (34 men, 27 women) with non-insulin-dependent diabetes mellitus (NIDDM) of whom 22 showed microalbuminuria and 39 normoalbuminuria. Data obtained in 31 non-obese and non-diabetic subjects were available for comparison. The plasma levels of fibrinogen (p < 0.02), prothrombin fragment 1 + 2 (p < 0.05), fibrin monomer (p < 0.0001), protein C antigen (p < 0.005), total protein S antigen (p < 0.02), soluble thrombomodulin (p < 0.005) and soluble E-selectin (p < 0.005) were significantly higher in diabetic patients than in healthy subjects. The plasma level of activated protein C-protein C inhibitor complex (7.4 +/- 3.8 vs 3.0 +/- 0.4 pmol/l) was significantly higher (p < 0.0001) and the anticoagulant response to exogenous thrombomodulin (23.4 +/- 2.6 vs 35.3 +/- 3.0 ng/ml) was markedly lower (p = 0.005) in all diabetic patients than in healthy subjects. Cases with microalbuminuria presented low plasma levels of activated protein C-protein C inhibitor complex (5.5 +/- 0.6 vs 8.6 +/- 0.7 pmol/l, p < 0.05) and significantly decreased values of the anticoagulant response to exogenous thrombomodulin (16.5 +/- 2.9 vs 23.4 +/- 2.6%, p = 0.03) as compared to those with normoalbuminuria. The present study suggests that the hyper-coagulable state in NIDDM is associated with an increased activation of protein C but with a poor plasma reactivity to the anticoagulant effect of thrombomodulin.

Adult↗
PubMed1996

beta 2-Glycoprotein I modulates the anticoagulant activity of activated protein C on the phospholipid surface.

The objective of this study was to determine whether beta 2-glycoprotein I (beta 2GPI) has procoagulant activity by inhibiting the anticoagulant activity of activated protein C (APC). beta 2GPI inhibited significantly the APC-catalyzed inactivation of factor Va in an assay using factor V-deficient plasma and physiological levels of protein S and factor Va. This inhibitory effect was diminished by the addition of increasing concentrations of phospholipids, suggesting that beta 2GPI competitively inhibits the binding of APC to the phospholipid surface. beta 2GPI inhibited weakly factor Va- and phospholipid-dependent prothrombinase activity at concentrations similar to those to inhibit APC activity. The depletion of beta 2GPI from plasma led to only a slight shortening of the diluted Russell's viper venom-dependent clotting time, but to a strong and significant potentiation of the anticoagulant activity of APC. These results suggest that under certain physiological conditions beta 2GPI has procoagulant property by inhibiting the phospholipid-dependent APC anticoagulant activity.

Anticoagulants↗
PubMed1996

Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus.

The salivary glands of the blood sucking insect, Rhodnius prolixus, have an anticoagulant, prolixin-S, which was reported as a specific inhibitor of intrinsic coagulant pathway. Prolixin-S was purified from the salivary glands extract of Rhodnius prolixus by gel filtration and anion exchange HPLC by assaying prolongation of activated partial thromboplastin time (APTT). The isolated protein specifically inhibited factor IXa-catalyzed activation of factor X in the presence of Ca2+ and phospholipids irrespective of the presence or absence of factor VIIIa. The anticoagulant factor had red color and a specific absorbance peak at 402 nm and thus it was identified as a heme protein. A Rhodnius prolixus salivary gland cDNA library was prepared, screened with an antibody against prolixin-S and its complete cDNA sequence was determined. cDNA and deduced amino acid sequences showed that prolixin-S is a novel anticoagulant of 19,922 Da, which has no sequence homology with any other anticoagulant reported so far.

Amino Acid Sequence↗
PubMed1995

Characterization of the binding of factor Xa to fibrinogen/fibrin derivatives and localization of the factor Xa binding site on fibrinogen.

The binding of human factor Xa to fibrinogen and its derivatives was characterized. Factor Xa bound to immobilized fibrin with a concentration at half-maximal binding (C50) of 100 nM. The 4-carboxyglutamic acid (Gla) domain of factor Xa is important in factor Xa binding to fibrin monomer, based on the following observations; the binding requires Ca2+; Gla-domain-lacking factor Xa could not bind to fibrin; factor Xa binding was significantly reduced by prior treatment of factor Xa with factor IX/factor-X-binding protein from the venom of Trimeresurus flavoviridis which specifically binds to the Gla domain of human factors IX and X. Factor Xa also bound to fibrinogen, fibrinogen degradation products (FDP)-D and FDP-E, with a similar affinity (C50 = 75-131 nM). In a solution-phase equilibrated binding assay, approximately 0.76 mol factor Xa bound to 1 mol fibrinogen with a dissociation constant of 180 nM. The binding of 125I-labeled factor Xa to the fibrin monomer was inhibited markedly by unlabeled factor Xa, but only slightly by thrombin, suggesting that the binding site of factor Xa on fibrin monomer differs from that of thrombin. We localized the binding site of factor Xa on fibrinogen: factor Xa bound strongly to the A alpha chain, but weakly to the B beta and gamma chains of fibrinogen. The A alpha chain was then digested with lysyl endopeptidase and separated by reverse-phase HPLC. Among resulting peptides, factor Xa bound specifically to a peptide corresponding to residues Asp82-Lys123 of the A alpha chain. This factor-Xa-binding site is located in the boundary between the central E domain and the terminal D domain of fibrinogen and is apparently distinct from the reported thrombin-binding site.

Binding Sites↗
PubMed1995

cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom.

The complete amino acid sequence of ecarin is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland cDNA library of Kenyan Echis carinatus. The cDNA sequence with 2379 base pairs encodes an open reading frame of 616 amino acids with a remarkable sequence homology to the putative precursor protein of trigramin from Trimeresurus gramineus venom (61% identity) and a large hemorrhagin, jararhagin, from the pit viper Bothrops jararaca venom (62% identity). Thus, ecarin, as well as jararhagin and trigramin, is translated as a precursor protein, which may be processed posttranslationally. The ecarin proprotein has a "cysteine switch" motif (-Pro-Lys-Met-Cys-Gly-Val-) similar to that involved in the activation of matrix metalloproteinase zymogens. The processed mature protein consists of 426 amino acid residues (residues 191-616), showing the strongest sequence similarity with that of Russell's viper venom factor X activator (RVV-X) heavy chain (64% identity). Like RVV-X heavy chain, ecarin contains metalloproteinase, disintegrin, and cysteine-rich domains. The metalloproteinase domain has a typical zinc-chelating sequence (-His-Glu-Xaa-Xaa-His-Xaa-Xaa-Gly-Xaa-Xaa-His-), as found in crayfish astacin. In the disintegrin domain of ecarin, the Arg-Gly-Asp sequence is replaced by Arg-Asp-Asp, as found in the disintegrin domains of RVV-X heavy chain (Arg-Asp-Glu) and a guinea pig sperm fusion protein, PH-30 beta (Thr-Asp-Glu). These findings show that while there are structural and evolutionary relationships among these proteins, each has a unique functional activity.

Amino Acid Sequence↗
PubMed1995

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site.

Human blood coagulation factor X has two N-linked oligosaccharides at Asn39 and Asn49 residues and two O-linked oligosaccharides at Thr17 and Thr29 residues in the region of the factor X activation peptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn39 and Asn49 residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri- and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac alpha 2-->6 residue. In addition, the structures of O-linked oligosaccharides at Thr17 and Thr29 residues were suggested to be disialylated Gal beta 3GalNAc sequences by their component analyses.

Asparagine↗
PubMed1995

Crystallization and preliminary X-ray study of H2-proteinase from the venom of Trimeresurus flavoviridis.

H2-proteinase, a non-hemorrhagic metalloproteinase from the venom of Trimeresurus flavoviridis, has been crystallized by vapor diffusion from solutions containing ammonium sulfate. The crystals belong to the tetragonal space group, P41212 or P43212, with unit cell dimensions of a = b = 77.8 A and c = 82.3 A. The asymmetric unit contains one protein molecule. Diffraction data for a native crystal were collected up to 2.0 A resolution.

Animals↗
PubMed1994

The thrombin receptor transmits signals through a 33-kDa protein kinase in human platelets.

In order to examine a possible role of protein kinases in the signal transduction of platelet activation, thrombin-stimulated human platelets were analyzed for protein kinase activity with a denaturation/renaturation method. Treatment of platelets with thrombin resulted in a rapid activation of a 33-kDa protein kinase (PK33) using casein as an in vitro substrate. The concentration of thrombin to activate PK33 was proportional to that required to induce platelet aggregation. PK33 was also activated by a thrombin receptor agonist peptide, but not by hirudin-treated or diisopropylphosphate-inactivated thrombin. Phosphoamino acid analysis showed that PK33 is a serine/threonine kinase. Comparative analysis using specific substrate and inhibitors revealed that PK33 is distinct from casein kinase I, casein kinase II, P34cdc2, and mitogen activated protein kinase. These findings suggest that platelet activation mediated by thrombin receptor requires PK33 activation.

Blood Platelets↗
PubMed1994

Receptor-mediated endocytosis of thrombin-antithrombin III complex by the human monocytoid cell line U937.

Receptor-mediated endocytosis of thrombin-antithrombin III (TAT) complex was characterized with the monocytoid cell line U937. At 4 degrees C, U937 cells bound TAT but not antithrombin III in a rapid, specific, saturable, and reversible manner. [125I]TAT binding to U937 cells was not inhibited by antithrombin III and hardly by thrombin, but 75% inhibited by a 50 molar excess of the unlabeled TAT. There were about 79,000 binding sites/cell (Kd = 68 nM). The TAT receptor on U937 cells may be distinct from the serpin-enzyme complex (SEC) receptor, since a synthetic peptide corresponding to the recognition sequence of SEC receptor did not compete with TAT binding to the cells. Receptor-bound TAT was internalized into the cells after switching the temperature from 4 to 37 degrees C and thereafter degraded in lysosomes.

Amino Acid Sequence↗
PubMed1994

The role of human factor X activation peptide in activation of factor X by factor IXa.

We studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5 mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5 mM EDTA, or in 8 M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa. Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.

Amino Acid Sequence↗
PubMed1994

Blood coagulation factor Xa interacts with a linear sequence of the kringle 2 domain of prothrombin.

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.

Amino Acid Sequence↗
PubMed1994

[Anticoagulant and fibrinolytic systems of the injured vascular endothelial cells].

Recent advances in determining anti-thrombogenic functions of vascular endothelial cells are reviewed. The following anticoagulant and fibrinolytic systems of endothelial cells are physiologically important; (1) Endothelial cell-derived metabolites including prostacyclin and nitric oxide (NO) support platelet inactivity. (2) Antithrombin III and tissue factor pathway inhibitor (TFPI) bound to heparin-like proteoglycans on endothelial cell membrane inhibit activated serine protease coagulation factors such as thrombin, factor Xa and factor VIIa-tissue factor complex. (3) Thrombomodulin converts thrombin from procoagulant into anticoagulant. Thrombin associated to thrombomodulin on endothelial cells activates protein C. Activated protein C in concert with protein S bound to endothelial cell membrane inactivates factors Va and VIIIa. (4) A receptor for both tissue plasminogen activator and plasminogen on endothelial cells provides an efficient plasmin generating system. Perturbation of these anti-thrombogenic systems of endothelial cells is caused by endotoxin (LPS), cytokines such as interleukin-1 and tumor necrosis factor (TNF), and risk factors for atherogenesis including lipoprotein(a) and homocysteine may result in arterial or venous thrombosis with subsequent development of atherosclerosis.

Animals↗