The Kringle V-Protease Domain Is a Fibrinogen Binding Region within Apo(a)

2001 ◽  
Vol 86 (11) ◽  
pp. 1229-1237 ◽  
Author(s):  
Song Xue ◽  
Edwin Madison ◽  
Lindsey Miles
Keyword(s):  
Binding Sites ◽  
Protease Domain ◽  
Binding Region ◽  
Molecular Weights ◽  
Fibrinogen Binding ◽  
Liver Cdna Library ◽  
Interaction Treatment ◽  
Dose Dependent Fashion ◽  
Affinity Interaction ◽  
Dose Dependent

SummaryLp(a) binds directly to fibrin and competes for the interaction of plasminogen with this substrate. This competition may play a role in the proatherothrombogenic consequences of high Lp(a) levels. Previous studies by us and others showed that apo(a) Kringle IV-10 competes for the interaction of Lp(a) with plasmin-treated fibrinogen. However, kringle IV-10 cannot account for the entire high affinity interaction of Lp(a) with fibrinogen. Therefore, we tested the hypothesis that the apo(a) kringle V protease-like domain (KV-PD) could interact with plasmin-treated fibrinogen. We cloned the apo(a) KV-PD region from a human liver cDNA library. Fusion apo(a) KV-PD was expressed in COS 7 cells and purified from the conditioned media. Western blotting of the apo(a) KV-PD protein revealed two bands migrating with apparent molecular weights of 45K and 48K. When fusion apo(a) KV-PD was treated with O-glycosidase and neuraminidase, the higher molecular weight band disappeared suggesting that the apo(a) KV-PD was O-glycosylated. Apo(a) KV-PD bound to plasmin-treated fibrinogen in a dose-dependent fashion. An EC50 of 3.9 ± 0.2 μM was determined for this interaction. Treatment of the apo(a) KV-PD with O-glycosidase did not significantly affect its ability to bind to plasmin-treated fibrinogen. In addition, apo(a) KV-PD competed for the binding of 125I-Lp(a) to plasmin-treated fibrinogen. An IC50 of 7.90 ± 0.95 μM was obtained. Our data suggest that the KV-PD of apo(a) shares binding sites on plasmin-treated fibrinogen with Lp(a) and also may participate in the interaction of the Lp(a) particle with plasmin-treated fibrinogen.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Effect of angiotensin II and deoxycorticosterone infusion on vascular angiotensin II receptors in rats

1984 ◽  
Vol 246 (4) ◽  
pp. H608-H614 ◽  
Author(s):  
E. L. Schiffrin ◽  
J. Gutkowska ◽  
J. Genest
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Binding Capacity ◽  
Plasma Renin ◽  
Intravenous Dose ◽  
Plasma Aldosterone ◽  
Ang Ii ◽  
Plasma Aldosterone Concentration ◽  
Dose Dependent Fashion ◽  
Dose Dependent

The effect of angiotensin II (ANG II) and deoxycorticosterone acetate (DOCA) on the density (Bmax) and affinity (Kd) of binding sites for 125I-ANG II was investigated in a particulate fraction prepared from rat mesenteric arteriolar arcades. Rats were infused with ANG II via Alzet osmotic minipumps at a dose of 200 ng X kg-1 X min-1 intraperitoneally or 60 and 200 ng X kg-1 X min-1 intravenously for 5 days. Bmax was 127 +/- 5 fmol/mg protein, and Kd was 0.8 +/- 0.1 nM in controls and was reduced significantly after the intraperitoneal infusion (111 +/- 10 fmol/mg) or the lower intravenous dose (111 +/- 9 fmol/mg), whereas after the higher intravenous dose Bmax did not change (144 +/- 14 fmol/mg). Kd was unaffected in all groups. Plasma renin activity (PRA) was reduced, and plasma ANG II increased in a dose-dependent fashion after ANG II infusion. Plasma aldosterone concentration increased only in the group infused with ANG II at 200 ng X kg-1 X min-1 intravenously (to 33.8 +/- 8.0 ng/dl from 11.6 +/- 3.4). In rats implanted subcutaneously with silicone rubber impregnated with DOCA, Bmax for 125I-ANG II was significantly increased (to 142 +/- 4 fmol/mg), whereas rats receiving 1% NaCl in their drinking water had no change in binding capacity, although PRA was lower in both groups. DOCA infusion, when combined with the intravenous dose of ANG II that reduced Bmax, antagonized this action of ANG II. DOCA infusion into sodium-depleted rats partially corrected the down-regulation of vascular ANG II receptors independent of changes in PRA.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

1996 ◽  
Vol 75 (01) ◽  
pp. 168-174 ◽  
Author(s):  
Shigeru Tokita ◽  
Morio Arai ◽  
Naomasa Yamamoto ◽  
Yasuhiro Katagiri ◽  
Kenjiro Tanoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent Fashion ◽  
Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

scholarly journals IMMU-09. CONCURRENT DEXAMETHASONE LIMITS THE CLINICAL BENEFIT OF IMMUNE CHECKPOINT BLOCKADE IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii106-ii106
Author(s):  
Bryan Iorgulescu ◽  
Prafulla Gokhale ◽  
Maria Speranza ◽  
Benjamin Eschle ◽  
Michael Poitras ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Immune Checkpoint ◽  
Nk Cell ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Innate Immune Responses ◽  
Dose Dependent Fashion ◽  
Dexamethasone Therapy ◽  
Clinical Correlate ◽  
Dose Dependent

Abstract BACKGROUND Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy. METHODS We evaluated the dose-dependent effects of dexamethasone when administered with anti-PD-1 and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 or CT-2A glioblastoma tumors, including analyses of intracranial tumors, draining lymph nodes, and spleen. Clinically, the effect of dexamethasone on survival was additionally evaluated in 181 consecutive IDH-wildtype glioblastoma patients treated with anti-PD-(L)1, with adjustment for relevant prognostic factors. RESULTS Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy decreased survival in a dose-dependent fashion and decreased survival following anti-PD-1 plus radiotherapy in both GL261 and immunoresistant CT-2A models. Dexamethasone quantitatively decreased T lymphocytes by reducing the proliferation while increasing apoptosis. Dexamethasone also decreased lymphocyte functional capacity. Myeloid and NK cell populations were also generally reduced. Thus, dexamethasone negatively affects both the adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed worse survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone use – regardless of dose – was the strongest predictor of poor survival (reference no dexamethasone; &lt; 2mg HR 2.28, 95%CI=1.41–3.68, p=0.001; ≥2mg HR 1.97, 95%CI=1.27–3.07, p=0.003). CONCLUSIONS Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for glioblastoma patients. Our preclinical analyses also suggest that dexamethasone’s detrimental effects are dose-dependent, suggesting that the lowest possible dose should be used for patients when dexamethasone use is unavoidable. Careful evaluation of dexamethasone use is warranted for neuro-oncology patients undergoing immunotherapy clinical trials.

Linkage of [Ca2+]i in single isolated D cells to somatostatin secretion induced by cholecystokinin

1996 ◽  
Vol 270 (6) ◽  
pp. G897-G901 ◽  
Author(s):  
J. DelValle ◽  
J. Wakasugi ◽  
H. Takeda ◽  
T. Yamada
Keyword(s):  
Channel Blocker ◽  
Gastric Fundus ◽  
Initial Transient ◽  
Somatostatin Secretion ◽  
Phospholipid Signaling ◽  
Dose Dependent Fashion ◽  
Direct Linkage ◽  
D Cells ◽  
Transient Elevation ◽  
Dose Dependent

The Ca2+/inositol phospholipid signaling cascade has been implicated in the mechanism by which cholecystokinin (CCK) stimulates gastric somatostatin release, but a direct linkage between intracellular events in gastric D cells and somatostatin secretion has not been established. To address this problem we developed a method for correlating somatostatin release with the measurement of intracellular Ca2+ concentration ([Ca2+]i) in isolated D cells. Resting [Ca2+]i in single D cells was 100 +/- 5.7 nM (means +/- SE, n = 41), and CCK induced a rise in [Ca2+]i in a dose-dependent fashion, producing a maximal stimulatory effect (243 +/- 15% of control, n = 12) at a peptide concentration of 2 x 10(-8) M. The CCK-mediated increase in [Ca2+]i was biphasic, with a rapid, initial transient elevation followed by a sustained plateau. The rise in [Ca2+]i was accompanied by a concomitant increase in release of somatostatin-like immunoreactivity (SLI). Removal of extracellular Ca2+ had no effect on the initial transient elevation in [Ca2+]i induced by CCK but abolished both the sustained plateau in [Ca2+]i and the release of SLI. The selective CCK antagonist L-364, 718 (10(-7) M) inhibited the effects of CCK on both [Ca2+]i and SLI release. The nonspecific Ca2+ channel blocker NiCl2 (10(-3) M) and the L-type Ca2+ channel blocker nifedipine inhibited the sustained rise in [Ca2+]i and the release of SLI but left the initial transient increase in [Ca2+]i unaltered. These results indicate that CCK-stimulated release of SLI from D cells in the gastric fundus is linked to influx of extracellular Ca2+ via L-type Ca2+ channels.

11-Dehydrocorticosterone, a glucocorticoid metabolite, inhibits aldosterone action in toad bladder

1991 ◽  
Vol 261 (5) ◽  
pp. F873-F879 ◽  
Author(s):  
A. S. Brem ◽  
K. L. Matheson ◽  
J. L. Barnes ◽  
D. J. Morris
Keyword(s):  
Sodium Transport ◽  
Cell Layer ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Hydroxysteroid Dehydrogenase ◽  
Toad Bladder ◽  
Compound A ◽  
Dose Dependent Fashion ◽  
Epithelial Cell Layer ◽  
Dose Dependent

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) metabolizes glucocorticoid hormones and diminishes their ability to induce sodium transport. In these studies, we determined the location of this enzyme in toad bladder and assessed the biological role for its 11-dehydro end product. Employing a polyclonal antibody directed toward 11 beta-OHSD and immunofluorescence techniques, we located the enzyme in the epithelial cell layer of the toad bladder. Although corticosterone (10(-7) M) can partially suppress aldosterone (10(-7) M)-stimulated short-circuit current (SCC), a clear excess of corticosterone (10(-6) M) did not inhibit the aldosterone-induced induced (10(-8) M) rise in SCC (n = 6). The 11-dehydro product of corticosterone, 11-dehydrocorticosterone (compound A) added to the serosal bath suppressed aldosterone (10(-8) M) peak SCC (360 min) in a dose-dependent fashion reaching 46 +/- 5% of control values at 10(-5) M (n = 6; P less than 0.001). Compound A (10(-5) M) in the mucosal bath also was capable of partially inhibiting the peak aldosterone rise in SCC to 63 +/- 7% of control values with aldosterone at 10(-8) M (n = 6; P less than 0.01) and to 64 +/- 10% of control values with aldosterone at 10(-7) M (n = 9; P less than 0.01). Compound A alone at 10(-5) M did not have any effect on SCC. Isolated toad bladders were not able to transform compound A (at 10(-8) and 10(-5) M) back to corticosterone. Thus the 11-dehydro end product of 11 beta-OHSD (compound A) may play a biologic role by regulating a component of mineralocorticoid-induced sodium transport.

scholarly journals Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites

1991 ◽  
Vol 274 (2) ◽  
pp. 457-463 ◽  
Author(s):  
J J Calvete ◽  
J Arias ◽  
M V Alvarez ◽  
M M Lopez ◽  
A Henschen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Human Platelet ◽  
Binding Domain ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Gamma Chain ◽  
Fibrinogen Binding ◽  
Glycoprotein Iiia ◽  
Membrane Bound

The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD-containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N-terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen-binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex.

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