In Vitro Inhibition of Blood Coagulation by Tripeptide Aldehydes - A Retrospective Screening Study Focused on the Stable D-MePhe-Pro-Arg-H · H2SO4

SummaryA series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.

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Inhibition of Adenosine Triphosphatases in Vitro by Silver Nitrate and Silver Sulfadiazine

Journal of the American College of Toxicology ◽

10.3109/10915818409009071 ◽

1984 ◽

Vol 3 (1) ◽

pp. 37-42 ◽

Cited By ~ 3

Author(s):

B. R. Nechay ◽

J. P. Saunders

Keyword(s):

Silver Nitrate ◽

Aqueous Suspension ◽

Human Kidney ◽

Silver Sulfadiazine ◽

Constant Concentration ◽

Inhibitory Potency ◽

Adenosine Triphosphatases ◽

Tissue Homogenates ◽

Atpase Inhibition

Inhibition of adenosine triphosphatase (ATPase) by silver nitrate (AgNO3) in vitro was studied in microsomal fractions or tissue homogenates of canine brain and kidney and human kidney. In microsomal fractions, AgNO3 was an indiscriminate inhibitor of ouabain-sensitive (Na+ + K+ ATPase) and ouabain-insensitive (Mg2+ ATPase) activities, with 50% inhibition obtaining at concentrations on the order of 10–7 to 10–6 M. Changing the concentrations of Na+, K+, H+, Mg2+, and ATP did not alter the fractional inhibition of Na+ + K+ ATPase by a constant concentration of AgNO3. An aqueous suspension of silver sulfadiazine had an inhibitory potency similar to AgNO3. It was concluded that silver gives a different pattern of Na+ + K+ ATPase inhibition than other metallic inhibitors of the enzyme so far examined.

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Influence Of Elastase On Blood Coagulation Factors: Studies In Vitro, In Rats And In Göttinger Miniatur Pigs

10.1055/s-0038-1653045 ◽

1981 ◽

Author(s):

U Kasten ◽

U Artmann ◽

T Kaethner ◽

H Burchardi ◽

H Köstering

Keyword(s):

Blood Coagulation ◽

Coagulation Factors ◽

Bleeding Complications ◽

Thrombin Time ◽

Normal Range ◽

Blood Coagulation Factors ◽

Acute Respiratory Insufficiency ◽

Coagulation Tests ◽

Antifibrinolytic Drugs

The influence of blood coagulation factors in pat. with acute respiratory insufficiency of adults, especially of the so called “pancreatitis lungs” is still unknown. In order to find out the effect of elastase, possibly activated by trypsin in pat. with acute pancreatitis, on blood coagulation factors, we performed some studies. In vitro elastase induces in plasma and blood in correlation to the dosages Enhancement of thrombingeneration in the TGT, a shortening of PTT, Thrombin time and of r- and k-time in the TEG, a loss of fibrinogen and an increase of fibrinmono-mercomplexes. In another study, elastase (960 U/ kg b.w.) was injected intravenously in rats. 30 min. later there was found a loss of fibrinogen, number of platelets, Prothrombin and a prolongation of PTT and Thrombin time and an increase of fibrinomonomercomplexes, especially in these rats, which received beside elastase Kalikreininhibitors or antifibrinolytic drugs. After repeated injections (3 times within 30 h) we found histomorpholgically thrombi as well as bleeding complications. In another study we performed (150 min) an infusion of elastase (333 U/kg b.w./h) to 9 pigs. We determined a loss of fibrinogen of platelets, of F. II, F. VII and F. XIII, a prolongation of PTT. F. VIII and F. V remained within the normal range But there was found an enhancement of Thrombin generation in the TGT, too. Compariening the results of blood coagulation tests and of histomorphological findings, elastase induced a DIC. We have to discuss their influence on ARIA and “Pancreatic lungs”.

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BIOCHEMICAL STUDIES ON CHLORPROMAZINE: 1. THE EFFECT OF CHLORPROMAZINE ON RESPIRATORY ACTIVITY OF ISOLATED RAT BRAIN CORTEX

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o57-130 ◽

1957 ◽

Vol 35 (12) ◽

pp. 1135-1144 ◽

Cited By ~ 27

Author(s):

O. Lindan ◽

J. H. Quastel ◽

S. Sved

Keyword(s):

Plasma Proteins ◽

Brain Cortex ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Acid Dyes ◽

Highly Sensitive ◽

Binding Power ◽

The Brain

Chlorpromazine exerts a progressive inhibitory activity (at 0.3–0.6 mM) on the respiration of brain cortex in presence of either glucose, fructose, pyruvate, or L-glutamate. A similar progressive inhibition occurs with other phenothiazine derivatives such as methylene blue and phenergan. However, chlorpromazine does not inhibit oxygen uptake in the presence of succinate. Potassium-stimulated respiration is highly sensitive to chlorpromazine, as it is markedly diminished by 0.2 mM concentration of the drug, a concentration which does not affect the unstimulated respiration. The increased inhibition of potassium-stimulated respiration is only clearly seen during the early part of the experiment.Chlorpromazine is bound by tissue constituents. At a constant concentration of chlorpromazine (0.6 mM), its inhibitory effect on cortical respiration may be abolished by markedly increasing the amount of tissue present. The inhibitory effect of chlorpromazine may be diminished by addition of plasma proteins (αβ-globulin) or by addition of heated homogenized brain, liver, or kidney. No binding occurs with polyglutamic acid, ribonucleic, and deoxyribonucleic acids, but binding does occur with certain acid dyes such as trypan red. Trypan red may be used to immobilize free chlorpromazine. When the latter drug is absorbed, however, by the nervous tissue, the addition of trypan red has no effect on the metabolic inhibitions brought about by the absorbed chlorpromazine.It is concluded that chlorpromazine resembles a large variety of narcotics and anaesthetics in its marked inhibitory effects on potassium-stimulated respiration of the brain. Its action, in vitro, however, differs from that of the narcotics in bringing about progressive, apparently irreversible, inhibitions and in its high binding power with tissue proteins. Such apparently irreversible inhibition is consistent with the conclusion that the drug, after combination with the tissue, gradually diffuses into the cell bringing about metabolic inhibitions.

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A Novel Congenital Bleeding Disorder Related to High Plasma Heparin-Like Anticoagulant Activity.

Blood ◽

10.1182/blood.v106.11.4074.4074 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4074-4074

Author(s):

Zhaoyue Wang ◽

Haiyen Yang ◽

Xia Bai ◽

Wei Zhang ◽

Changgeng Ruan

Keyword(s):

Ex Vivo ◽

Anticoagulant Activity ◽

Coagulation Factors ◽

High Plasma ◽

Bleeding Disorder ◽

Normal Plasma ◽

Normal Ranges ◽

Coagulation Tests ◽

Plasma Heparin

Abstract Heparin or heparin-like compounds present in human plasma in minute amounts. It has been reported that a very few patients with such diseases as plasma cell neoplasms, acute monoblastic leukemia and acquired immune deficincy syndrome have an increased plasma heparin-like anticoagulant activity. Recently, we found a 10-year-old girl who was physically and developmentally normal, but had recurrent episodes of prolonged bleeding and hematoma starting in her early childhood, which could be stopped by transfusion of fresh frozen plasma or prothrombin complex concentrate. The coagulation tests of her plasma were regularly repeated since she was 2 years old, and always revealed a markedly prolonged APTT (61.8–104 seconds, normal 28–40 seconds) and TT (36–50.1 seconds, normal 14–21 seconds), and a slightly prolonged PT (15.9–25 seconds, normal 11–14.5 seconds). Fibrinogen, prothrombin and other coagulation factors as well as anticoagulant and fibrinolytic systems were all normal. The results of immunologic measurements were either negative or within normal ranges. Treatment of the patient’s plasma in vitro with either protamine or heparinase could completely normalize the coagulation abnormalities, but not with normal plasma. The anticoagulant activity of her plasma corresponded to 0.2 heparin U/mL when measured by a TT assay using normal plasma as substrate and standardized with porcine heparin. Her plasma heparin concentration was 0.22 heparin U/mL when measured using a colometric assay. In ex vivo study, the abnormal coagulation tests could effectively be corrected when the patient was intravenously administed with protamine. Considering these characteristic laboratory features of the patient, we suppose it would probably represent a novel congenital bleeding disorder related to high plasma heparin-like anticoagulant activity which, to our knowledge, had not been described before.

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Excretion in the House Cricket (Acheta Domesticus): Stimulation of Diuresis by Tissue Homogenates

Journal of Experimental Biology ◽

10.1242/jeb.129.1.63 ◽

1987 ◽

Vol 129 (1) ◽

pp. 63-81 ◽

Cited By ~ 2

Author(s):

JEFFREY H. SPRING ◽

SHELIA R. HAZELTON

Keyword(s):

Lethal Dose ◽

Fluid Secretion ◽

Sodium Concentration ◽

Inhibitory Effect ◽

Acheta Domesticus ◽

Malpighian Tubules ◽

Secretion Rate ◽

Tissue Homogenates ◽

Extreme Sensitivity

1. A new method is described for maintaining cricket Malpighian tubules in vitro. Warmed, oxygenated saline is circulated rapidly past the tubules, while the secreted urine is collected under oil for analysis. This technique allows the cricket tubules to be observed and manipulated for extended periods (6 h), in contrast to their short life (>1 h) using conventional methods. 2. Cricket tubules show extreme sensitivity to oxygen deprivation, such that 15 min of anoxia represents the median lethal dose (LD50) for in vitro preparations. 3. Homogenates of corpus cardiacum (CC) cause the rate of fluid secretion by the tubules to double. The maximum stimulation is dose-dependent over the range 0.01 to 1.0 CC. Homogenates of brain and other ganglia show much smaller stimulatory effects (0.01-0.02 CC-equivalents). Cyclic AMP mimics the increase in secretion rate, but has an inhibitory effect on the smooth muscle of the ureter. 4. Control preparations maintain a urine osmotic pressure (OP) that is hyperosmotic to the bath by 5–10 mosmol l−1. CC homogenate produces a decrease in urine OP to 10–12 mosmol l−1 hypo-osmotic to the bath. This suggests that active solute reabsorption is occurring in the lower tubule or ampulla. 5. Stimulation by CC homogenate increases the urine potassium concentration slightly less than two-fold, whereas the sodium concentration increases by a maximum of five-fold and remains at a higher concentration than potassium throughout the experiment. Tubule secretion rate is drastically inhibited in nominally sodium-free saline.

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Coagulation Studies in a Case of Hageman Trait

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654919 ◽

1961 ◽

Vol 05 (02) ◽

pp. 187-200 ◽

Cited By ~ 3

Author(s):

E. A Loeliger ◽

A Hensen

Keyword(s):

High Activity ◽

Blood Coagulation ◽

Fibrinolytic Activity ◽

Coagulation Factors ◽

Normal Rate ◽

Hageman Factor ◽

Haemorrhagic Diathesis ◽

Anticoagulant Property

SummaryAfter a brief review of the data concerning the cases of Hageman trait hitherto reported in the literature, a patient with severe Hageman factor (HF) “deficiency” (HF-activity below 0.05% of normal) is described. The patient has no haemorrhagic diathesis.In vitro, intrinsic coagulability and fibrinolytic activity are grossly disturbed. On the basis of a diminished fibrinolytic activity in vivo, the rather high activity of several coagulation factors in patient’s plasma could be explained.Thromboplastin formation, once initiated, is normal as to rate; the amount of thromboplastin formed is possibly slightly diminished. Prothrombin consumption is normal. These findings are in agreement with a normal rate ot clot formation, as measured by means of thrombelastography.Normal HF seems to be an initiator of blood coagulation only and not an activator or a substrate involved in thromboplastin formation.The weak anticoagulant property of Hageman trait plasma, described by several authors, is not necessarily due to an inhibitor; it can be explained by the assumption that, in Hageman trait, HF shows normal glass adsorbability and only a very deficient glass activation.

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Anticoagulant Effect of Sulphated Poly/Vinyl Alcohol-Acrylic Acid/Copolymers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661690 ◽

1986 ◽

Vol 56 (03) ◽

pp. 397-400 ◽

Cited By ~ 7

Author(s):

Raymund Machovich ◽

Miklós Nagy ◽

Judit Györgyi-Edelényi ◽

Katalin Csomor ◽

István Horváth

Keyword(s):

Acrylic Acid ◽

Blood Coagulation ◽

Vinyl Alcohol ◽

Blood Clotting ◽

Antithrombin Iii ◽

Inhibitory Effect ◽

Poly Vinyl Alcohol ◽

Direct Inhibitory Effect ◽

Vitro Effect

SummaryCopolymers of poly/vinyl alcohol-acrylic acid/ with various content of sulphate and carboxyl groups have been synthetized and tested for their in vitro effect on blood coagulation. The results indicate that the sulphated copolymers display an inhibitory effect but there is a requirement in the charged groups of about 20% in the molecqle to possess effective anticoagulation. The biochemical mechanism of their actions is complex, i.e. the inhibition of blood clotting is a consequence of both (i) the accelerated inactivation rate of thrombin by antithrombin-III and (ii) a direct inhibitory effect on the thrombin-fibrinogen reaction. Moreover, additional effects may occur on other blood coagulation enzymes than thrombin, depending on the chemical composition of the copolymers.

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In Vivo Studies On The Inhibition Of Coagulation By A Heparin Analogue

10.1055/s-0038-1652526 ◽

1981 ◽

Author(s):

U Schmitz-Huebner ◽

F Asbeck ◽

J van de Loo

Keyword(s):

Plasma Sample ◽

Anticoagulant Activity ◽

Inhibitory Effect ◽

In Vivo Studies ◽

At Iii ◽

In Vivo Effects ◽

Higher Doses ◽

Free Plasma

SSHA, a semi-synthetic heparin analogue belonging to the chondroitin family, was reported to induce considerable anti-Xa activity in vivo being practically inactive in vitro. In a study designed to elucidate further the in vivo effects of this drug, SSHA and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six volunteers on separate occasions over a period of three days in a cross-over trial. Before injection and 2,4,6,8 hours afterwards, the heparin-like activity was measured by means of the APTT, the anti -Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical, due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induce proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). In an attempt to identify the mediator involved in the anticoagulant activity induced by SSHA in vivo, antithrombin III AT III) was removed from a plasma sample of one the subjects obtaining SSHA injections by immunosorption using Sepharose IVb coupled with antibodies against AT III. The AT III free plasma obtained was found to be devoid of heparin-like activity in the anti-Xa clotting test but it maintained its anticoagulant activity in the APTT assay. When purified AT III was added to this plasma its anti-Xa activity was largely restored. In conclusion, the inhibitory effect of SSHA on coagulation seems to involve at least two mechanisms: a direct one which does not depend on AT III and an indirect one, induced in vivo and mediated by AT III.

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THE EFFECT OF THE VENOM OF THE ORIENTAL HORNET ON COAGULATION FACTORS

10.1055/s-0038-1644338 ◽

1987 ◽

Author(s):

A Kornberg ◽

S Kaufman ◽

L Silber ◽

J Ishay

Keyword(s):

Human Plasma ◽

Degradation Products ◽

Anticoagulant Activity ◽

Coagulation Factors ◽

Plasma Fibrinogen ◽

Thrombin Time ◽

Clotting Factors ◽

Viii And Ix

The extract from the venom sac of Vespa orientalis (VSE) inactivates exogenous and endogenous thromboplastin (Joshua and Ishay, Toxicon, 13:11-20,1975). The prolongation of both prothrombin time (PT) and recalcification time suggests inactivation of other factors. The aim of the present study is to investigate the effect of VSE on clotting factors. A lyophilized VSE with protein concentration of 5 mg/ml was used. Studies were performed in vitro with human plasma and in vivo in cats. Routine methods were employed for the assay of PT, activated tissue thromboplastin (APTT), thrombin time (TT), fibrinogen degradation products (FDP), fibrinogen and factors V,VII,VIII,IX,X. Human plasma was incubated with various concentrations of VSE (0,1,5,10,50,100 μg/ml) for 60 min and for various incubation times (0,5,15,30,+ 60,90,120 min) with 50 μg/ml VSE (n=8). 1 μg/ml VSE prolonged PT from 13.5 to 16 sec (p<0.05) and APTT from 62 to 180 sec. PT was maximal (17.7 sec) with 10 μg/ml and APTT (442 sec) with 50 μg/ml VSE. Factors V,VII,X decreased gradually from 94-105% to 11%,11% and 29% with 100 μg/ml VSE and VIII and IX to 1% even with 1 μg/ml VSE. After 5 min with constant concentration of VSE (50 μg/ml) PT was 14.9 sec (normal 13 sec) and APTT 165 sec (normal 54 sec). Both were maximal (17.5 and 298 sec) after 60 min. Factors VII and X decreased to 13% and 32% and VIII and IX to >1% after 60 min of incubation. Injection of 5 mg/kg VSE to cats (n=6-8) resulted in prolongation of PT from 9.4 to 11.2 sec and of APTT from 19.5 to 63 sec after 5 min. Both were maximal after 90 min (12.3 and 127 sec). Factors V,VII and X decreased from 100% to 7.6%, 13% and 37% and VIII and IX to 1% after 10 min. In all experiments TT and plasma fibrinogen were not affected and FDP were normal. Heating of VSE for 5 min at 80°C abolished completely the anticoagulant activity but dialysis for 24 hr at 4°C had no effect on it. The activity was eluted on Sephadex-25 both in void and post void volumes. The results show that VSE has a potent anticoagulant activity against various factors. Factors VIII and IX are markedly decreased. The effect on V, VII and X is moderate. Plasma fibrinogen is not affected. The nature and clinical significance of the anticoagulant activity merit further investigation.

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Synthesis, Docking, and In Vitro Anticoagulant Activity Assay of Hybrid Derivatives of Pyrrolo[3,2,1-ij]Quinolin-2(1H)-one as New Inhibitors of Factor Xa and Factor XIa

Molecules ◽

10.3390/molecules25081889 ◽

2020 ◽

Vol 25 (8) ◽

pp. 1889 ◽

Cited By ~ 1

Author(s):

Nadezhda Novichikhina ◽

Ivan Ilin ◽

Anna Tashchilova ◽

Alexey Sulimov ◽

Danil Kutov ◽

...

Keyword(s):

Anticoagulant Activity ◽

Factor Xa ◽

Coagulation Factor ◽

Coagulation Factors ◽

Protein Targets ◽

Ic50 Value ◽

Anticoagulant Drugs ◽

Factor Xia ◽

Derivatives Of

Coagulation factor Xa and factor XIa are proven to be convenient and crucial protein targets for treatment for thrombotic disorders and thereby their inhibitors can serve as effective anticoagulant drugs. In the present work, we focused on the structure–activity relationships of derivatives of pyrrolo[3,2,1-ij]quinolin-2(1H)-one and an evaluation of their activity against factor Xa and factor XIa. For this, docking-guided synthesis of nine compounds based on pyrrolo[3,2,1-ij]quinolin-2(1H)-one was carried out. For the synthesis of new hybrid hydropyrrolo[3,2,1-ij]quinolin-2(1H)-one derivatives, we used convenient structural modification of both the tetrahydro- and dihydroquinoline moiety by varying the substituents at the C6,8,9 positions. In vitro testing revealed that four derivatives were able to inhibit both coagulation factors and three compounds were selective factor XIa inhibitors. An IC50 value of 3.68 μM for was found for the best factor Xa inhibitor and 2 μM for the best factor XIa inhibitor.

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