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

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.

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Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655587 ◽

1964 ◽

Vol 12 (01) ◽

pp. 232-261 ◽

Cited By ~ 2

Author(s):

S Sasaki ◽

T Takemoto ◽

S Oka

Keyword(s):

Molecular Weight ◽

Dextran Sulfate ◽

Anticoagulant Activity ◽

Molecular Structures ◽

Severe Reaction ◽

Clinical Use ◽

Molecular Weights ◽

Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

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Anticoagulant Properties of a Green Algal Rhamnan-type Sulfated Polysaccharide and Its Low-molecular-weight Fragments Prepared by Mild Acid Degradation

Marine Drugs ◽

10.3390/md16110445 ◽

2018 ◽

Vol 16 (11) ◽

pp. 445 ◽

Cited By ~ 7

Author(s):

Xue Liu ◽

Peng Du ◽

Xiao Liu ◽

Sujian Cao ◽

Ling Qin ◽

...

Keyword(s):

Molecular Weight ◽

Anticoagulant Activity ◽

Sulfated Polysaccharide ◽

Low Molecular Weight ◽

Thrombin Time ◽

Acid Degradation ◽

Molecular Weights ◽

Green Algal

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1–MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1–MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1–MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1–MSP-F4 with molecular weights of 24–240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.

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Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments

Biochemical Journal ◽

10.1042/bj2770863 ◽

1991 ◽

Vol 277 (3) ◽

pp. 863-868 ◽

Cited By ~ 32

Author(s):

D Sömjen ◽

K D Schlüter ◽

E Wingender ◽

H Mayer ◽

A M Kaye

Keyword(s):

Parathyroid Hormone ◽

Bone Resorption ◽

Thymidine Incorporation ◽

Specific Activity ◽

Fold Increase ◽

Dependent Manner ◽

Equimolar Concentration ◽

Dose Dependent

We have found, in previous studies in vitro using skeletal derived cell cultures, that mid-region fragments of human parathyroid hormone (hPTH) stimulate [3H]thymidine incorporation into DNA and increase the specific activity of the brain-type isoenzyme of creatine kinase (CK). These changes occurred without an increase in cyclic AMP formation which is linked to bone resorption. In this study, we found that the mid-region fragment hPTH-(28-48) stimulated CK activity in diaphysis, epiphysis and kidney in a time- and dose-dependent manner, parallel to the effects of the whole molecule bovine (b)PTH-(1-84) and the fully active fragment hPTH-(1-34). The increase caused by hPTH-(28-48) at a dose of 1.25 micrograms/rat was not less than the 2-fold increase caused by a roughly equimolar concentration bPTH-(1-84). A significant increase was reached at 1 h after intraperitoneal injection in all cases. All three sequences of PTH caused an increase in [3H]thymidine incorporation into DNA in diaphysis and epiphysis, but not in kidney, 24 h after injection. A fragment further towards the C-terminal, hPTH-(34-47), was inactive compared with an equimolar concentration of the fragment hPTH-(25-39), which stimulated both CK activity and DNA synthesis. These results in vivo are in line with previous findings in vitro; they provide further support for the suggestion that mid-region fragments of the PTH molecule could be used to induce bone formation without incurring the deleterious effect of bone resorption.

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Cloned LYT-2+ cytolytic T lymphocytes destroy allogeneic tissue in vivo.

Journal of Experimental Medicine ◽

10.1084/jem.159.1.234 ◽

1984 ◽

Vol 159 (1) ◽

pp. 234-243 ◽

Cited By ~ 46

Author(s):

J D Tyler ◽

S J Galli ◽

M E Snider ◽

A M Dvorak ◽

D Steinmuller

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Cytolytic T Lymphocytes ◽

Transplantation Immunity ◽

Dose Dependent Fashion ◽

Dose Dependent

The long-accepted notion that alloimmune cytolytic T cells (CTL) mediate transplantation immunity has recently been called into question. In order to ascertain directly whether alloimmune CTL can mediate destruction of foreign tissue, we tested the ability of mouse CTL expanded as cloned populations in vitro to destroy allogeneic skin in vivo. The results of these studies prove unequivocally that cloned Lyt-2+ CTL can perform this task in an immunologically specific, H-2-restricted, and dose-dependent fashion.

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Effect of Platelet-activating Factor onin vitroandin vivoInterleukin-6 Production

Mediators of Inflammation ◽

10.1155/s0962935194000384 ◽

1994 ◽

Vol 3 (4) ◽

pp. 281-285 ◽

Cited By ~ 3

Author(s):

B. Pignol ◽

T. Maisonnet ◽

P. Guinot ◽

J. M. Mencia-Huertac ◽

P. Braquet

Keyword(s):

Platelet Activating Factor ◽

Similar Extent ◽

Rat Serum ◽

Mouse Fibroblasts ◽

Dose Dependent Fashion ◽

The One ◽

Dose Dependent ◽

Peak Increase

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1β and polyriboinositic/polyribocytidylic (poly I–C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 μM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 μM PAF, the peak increase (60.1 ± 19.4 U/ml) was similar to that induced by 50 μg/ml poly I–C (60.0 ± 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1β (3.8 ± 1.8 U/ml). The increase of 11-6 activity induced by 5 μM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 μM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 μM PAF. In addition, the IL-6 activity induced by 5 μM PAF was still observed when the specific PAF antagonist, BN 52021 (10 μM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAFin vitrois not receptor mediated. Thein vivoeffect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 μg/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 μg/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that thein vivoeffect is receptor mediated.

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CYCLOOXYGENASE INDIPENDENT PLATELET AGGREGATION:RELATION WITH ASPI RIN CONCENTRATION

10.1055/s-0038-1644828 ◽

1987 ◽

Author(s):

C Cordova ◽

F Violi ◽

D Praticò ◽

A Ghiselli ◽

C Alessandri ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Low Doses ◽

Dose Dependent Fashion ◽

Threshold Doses ◽

High Doses ◽

Dose Dependent

Low doses of aspirin (20 mg/day) were previously reported to be uneffective in preventing platelet aggregation (PA) induced by pairs of aggregating agents such as PAF and adrenalin.This was in part attributed to the inability of such treatment to inhibit lipo oxygenase-dependent PA.The latter can be observed in vitro in"aspl rinated"platelets stimulated with high quantities of aggregating -agents.The aim of this study was to evaluate if the lipooxygenase-dependent PA was influenced by aspirin in a dose-dependent fashion. PA was studied in platelet rich plasma (PRP)(Born's method) by using threshold doses of aggregating agents (TDA) such as PAF(4-75 nM),epinephrine(0.6-2 μM) and collagen(2-4 μg/ml).PA performed in PRP pretrated with 100μM aspirin was fully prevented;in the same samples thromboxane (Tx) A2 evaluated by its metabolite Tx B2 was almost absent.Increasing amount of PAF(20 fold TDA),epinephrine(20 fold TDA) and collagen (36 fold TDA) do aggregate"aspirinated"pla telets;similarly"aspirinated"platelets aggregate when stimulated-with a pair of aggregating agents (TDA of PAF+epinephrine).This phenomenon was not detected if platelets were incubated with higher amounts of aspirin (250-500 μM).The study suggests that aspirin could influence lipooxygenase-dependent PA.This hypothesis is sup ported by a research showing the aspirin inhibits dose-dependently platelet HETE formation.A further study is now in progress to eva luate the influence of high doses of aspirin on cyclooxygenase-i"n dependent PA in vivo.

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Antiangiogenesis Is Produced by Nontoxic Doses of Vinblastine

Blood ◽

10.1182/blood.v94.12.4143 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4143-4155 ◽

Cited By ~ 188

Author(s):

Angelo Vacca ◽

Monica Iurlaro ◽

Domenico Ribatti ◽

Monica Minischetti ◽

Beatrice Nico ◽

...

Keyword(s):

Inflammatory Diseases ◽

Wide Spectrum ◽

Chorioallantoic Membrane ◽

Ultrastructural Level ◽

Matrix Metalloproteinase 2 ◽

Cell Functions ◽

Dose Dependent Fashion ◽

Dose Dependent

Abstract The effects of vinblastine (VBL) on endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, spreading on fibronectin (FN), secretion of matrix-metalloproteinase-2 (MMP-2) and MMP-9, and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. In vitro, at noncytotoxic doses (0.1, 0.25, 0.5, 0.75, and 1 pmol/L), VBL impacted all these functions, except secretion of MMPs, in a dose-dependent fashion. By contrast, proliferation of other primary cells such as fibroblasts and lymphoid tumor cells was not impacted. In vivo, VBL at 0.5, 0.75, and 1 pmol/L again displayed a dose-dependent antiangiogenic activity. Lack of cytotoxicity in vitro and in vivo was shown both morphologically, and also because the antiangiogenic effects were rapidly abolished when VBL was removed. Apoptosis was not induced. At the ultrastructural level, impairment of cell functions in vitro was associated with thin disturbance of the cytoskeleton, in the form of slight depolymerization and accumulation of microfilaments, which was equally reversible. Results suggest that VBL has an antiangiogenic component at very low, noncytotoxic doses, and that antiangiogenesis by VBL could be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases, Kaposi's sarcoma, and cancer.

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Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives

Blood ◽

10.1182/blood-2010-06-288522 ◽

2011 ◽

Vol 117 (6) ◽

pp. 2054-2060 ◽

Cited By ~ 27

Author(s):

Elsa P. Bianchini ◽

Judicael Fazavana ◽

Veronique Picard ◽

Delphine Borgel

Keyword(s):

Anticoagulant Activity ◽

Fold Increase ◽

Glycosylation Site ◽

Dependent Manner ◽

Heparin Derivatives ◽

Dose Dependent ◽

Heparin Derivative ◽

In Vivo Administration

Abstract Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.

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A Heparin Analogue that Releases Endogenous Heparan Sulphate

10.1055/s-0039-1687056 ◽

1979 ◽

Author(s):

D.P. Thomas ◽

T.W. Barrowcliffe ◽

E.A. Johnson ◽

J. Stocks ◽

J. Dawes ◽

...

Keyword(s):

Lipoprotein Lipase ◽

Specific Activity ◽

Anticoagulant Activity ◽

Heparan Sulphate ◽

Factor Xa ◽

Fold Increase ◽

Endothelial Lining ◽

Drug Induced

Heparan sulphate (HS), a near-relative of heparin, but with much less anticoagulant activity in vitro, is bound to cell surfaces. We examined HS isolated from porcine intestinal mucosa, and found that although the material had low anticoagulant activity by APTT, it had a marked effect in anti-Factor Xa clotting assays, giving anti-Xa/APTT ratios of approximately 4:1 in terms of specific activity. In crossed immunoelectrophoresis experiments, HS bound to antithrombin III, but at higher concentrations than heparin. A heparin analogue (a polysulphated chondroitin), while virtually inactive in vitro, nevertheless when administered s.c. to man potentiated the effect of anti-Factor Xa to an extent comparable to that produced by low-dose heparin, but with an anti-Xa/APTT ratio of 4:1. The analogue also produced a marked release of lipoprotein lipase and a four-fold increase in the level of circulating PF4, as measured by radioimmunoassay. The anti-Xa and APTT effects of HS in vitro are very similar to those produced by the analogue in vivo, and it is suggested that the analogue releases not only lipoprotein lipase and PF4, but also HS. The drug-induced release of an endogenous glycosamino-glycan (probably from the endothelial lining of vessels) with anti-Xa activity may represent a fruitful approach to the prophylaxis of venous thrombosis.

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Role of 25-hydroxyvitamin D3 dose in determining rat 1,25-dihydroxyvitamin D3 production

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.258.5.e780 ◽

1990 ◽

Vol 258 (5) ◽

pp. E780-E789 ◽

Cited By ~ 1

Author(s):

R. Vieth ◽

K. McCarten ◽

K. H. Norwich

Keyword(s):

Specific Activity ◽

Metabolic Clearance ◽

Dihydroxyvitamin D3 ◽

Hydroxyvitamin D ◽

Dihydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Dose Dependent

To understand the relationships among 1) the dose of 25-hydroxyvitamin D [25(OH)D] in vivo, 2) the activity of 1-hydroxylase in renal mitochondria, and 3) the production of 1,25-dihydroxyvitamin D [1,25(OH)2D] in vivo, we gave rats different chronic or acute doses of 25-hydroxyvitamin D3 [25(OH)D3]. We followed the metabolism of intracardially administered [25-hydroxy-26,27-methyl-3H]cholecalciferol [25(OH)[3H]D3] for 24 h before killing by measuring extracts of serum by chromatography. Specific activity of 1-hydroxylase in kidney was measured at death. In rats given 0-2,000 pmol 25(OH)D3 chronically by mouth, there was a dose-dependent decline in the percent of serum radioactivity made up of 1,25-dihydroxy-[26,27-methyl-3H]cholecalciferol [1,25(OH)2[3H]D3] as well as a decline in mitochondrial 1-hydroxylase, and these correlated significantly (r = 0.83, P less than 0.001). Serum %1,25(OH)2[3H]D3 in this experiment ranged from 0.8 to 42%. A small part of this range could be accounted for by a faster metabolic clearance rate (MCR) of 1,25(OH)2D3 from rats supplemented with 25(OH)D3 (MCR, 2.12 +/- 0.10 ml/min) compared with rats restricted in vitamin D (MCR, 0.94 +/- 0.06 ml/min, P less than 0.001). The activity of 1-hydroxylase was by far the major factor determining serum %1,25(OH)2[3H]D3. When different acute doses of 25(OH)D3 were given to rats with identical specific activities of 1-hydroxylase, the resulting 1,25(OH)2D3 concentrations in serum correlated with the 25(OH)D3 dose (r = 0.99, P less than 0.001). We conclude that the behavior of 1-hydroxylase in vivo is analogous to the classic behavior in vitro of an enzyme functioning below its Michaelis constant (Km). The amount of 1-hydroxylase present in renal mitochondria determines the fraction (not simply the quantity) of 25(OH)D metabolized to 1,25(OH)2D3 in vivo.

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