Comparative Studies Of Purified Poloxamer 188 Using ClotBased and Viscoelastic Measurements Of Coagulation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4770-4770
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Schuharazad Abro ◽  
Martin Emanuele
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Maximum Amplitude ◽  
Fibrin Clot ◽  
Clotting Time ◽  
Poloxamer 188 ◽  
Biochemical Systems ◽  
High Concentrations ◽  
Adhesive Interactions ◽  
Clinically Significant

Background Poloxamer 188 (P188; Mast Therapeutics, Inc.) is a surface-active, non-ionic block copolymer which binds to hydrophobic surfaces on damaged cells improving membrane hydration and lowering adhesion and viscosity.  It is known to improve microvascular function in various pathologic states.  Currently, this agent is under investigation in a phase 3 clinical trial for treatment of sickle cell disease patients experiencing acute vaso-occlusive crisis.  The effect of P188 on blood coagulation and platelet function has been evaluated in several clinical trials where no clinically significant effect has been observed.   In these trials, coagulation studies were based on standard clot based methods (e.g., PT and aPTT) and did not include viscoelastic measurements such as thromboelastography (TEG).   Given P188 alters viscosity, we compared the effect of this agent using various clotbased, chromogenic and viscoelastic measurements of blood coagulation. Materials and Methods Whole blood activated clotting time studies were carried out in groups of healthy individuals (n=10) at a concentration range of 1.872-15.0mg/mL. TEG analysis on native and citratedwhole blood was carried out on TEG 5000 (Haemoscope Corp, Niles, IL) at concentrations of 0-0.45 mg/mL. The effect of P188 on normal plasma clotting parameters, such as PT and aPTT, was measured at a concentration range of 0-10 mg/mL. The effect of P188 on thrombin-induced clot formation was investigated using a fibrinokinetic method. The effect of P188 on thrombin generation was measured using the fluormetric method (Technoclone, Vienna, Austria). The anti-protease effects of P188 were studied using chromogenic substrate methods using isolated biochemical systems. Results At concentrations up to 10 mg/mL, P188 did not produce any modification of Celiteactivated clotting time (Celite-ACT).  At all concentrations the Celite-ACT values remained comparable to saline (138-140 sec for P188 vs. 140 sec for saline).  In the TEG analysis, P188 produced a concentrationdependent hypocoagulant effect in both native and citratedblood as evidenced by increased angle and shortening of maximum amplitude (MA).   In standard PT and aPTT tests, P188 did not produce any effect on the clotting profile at concentrations up to 20 mg/mL.  In fibrinokinetic studies, P188 produced an increase in the fibrin clot density and rate of fibrin polymerization. Discussion These studies demonstrate that even at very high concentrations, P188 does not produce an effect on whole blood clotting as measured by the Celite-ACT assay;   this result was confirmed in other standard assays.  Fibrinokinetic studies revealed an increase in the rate of fibrin formation and clot density.   However, at relatively low concentrations, P188 exhibited a hypocoagulant profile in TEG analysis.  The marked discordance between TEG and other coagulation tests suggest that P188’s effect on viscosity and adhesive interactions result in an artifact in TEG analysis and an incorrect indication of a hypocoagulant effect.  This effect may be due to the viscoelastic endpoint in the TEG assay. Further studies are needed to confirm this hypothesis. Disclosures: Fareed: Mast Therapeutics: Research Funding. Emanuele:Mast Therapeutics: Employment.

THE EFFECT OF EARLY BLOOD TRANSFUSION ON THE OUTCOME OF GASTROINTESTINAL HAEMORRHAGE

1987 ◽  
Author(s):  
S D Blair ◽  
S B Javanvrin ◽  
C N McCollum ◽  
R M Greenhalgh
Keyword(s):  
Blood Transfusion ◽  
Blood Coagulation ◽  
Whole Blood ◽  
Randomised Trial ◽  
Gastrointestinal Haemorrhage ◽  
Clotting Time ◽  
Prospective Randomised Trial ◽  
Coagulation Tests ◽  
To Receive ◽  
Upper Gastrointestinal

It has been suggested that mortality due to upper gastrointestinal haemorrhage may be reduced by restricting blood transfusion [1], We have assessed whether this is due to an anticoagulant effect in a prospective randomised trial.One hundred patients with severe, acute gastrointestinal haemorrhage were randomised to receive either at least 2 units of blood during the first 24 hours of admission, or no blood unless their haemaglobin was lessthan 8g/dl or they were shocked. Minor bleeds and varices were excluded As hypercoagulation cannot be measured using conventional coagulation tests, fresh whole blood coagulation was measured by the Biobridge Impedance Clotting Time (ICT). Coagulation was assessed at 24 hour intervals and compared to age matched controls with the results expressed as mean ± sem.The ICT on admission for the transfusion group (n=50) was 3.2±0.2 mins compared to 10±0.2 mins in controls. This hyper-coagulable state was partially reversed to 6.4±0.3 mins at 24 hours (p<0.001). The 50 allocated to receive no blood had a similar ICT on admission of 4.4±0.4 mins but the hypercoagulable state was maintained with ICT at 24 hours of 4.320.4 mins. Only 2 patients not transfused rebled compared to 15 in the early transfusion group (p<0.001). Five patients died, and they were all in the early transfusion group.These findings show there is a hypercoagulable response to haemorrhage which is partially reversed by blood transfusion leading to rebleeding

A Comparison of Inhibitors for Thrombin, FXa, FXIa, and FXIIa in Whole Blood Coagulation and Fibrinolysis Using Thromboelastography

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2256-2256
Author(s):  
Zhu Chen ◽  
Weizhen Wu ◽  
Yiming Xu ◽  
Martin L Ogletree ◽  
Andrew S Plump ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Fibrinolytic Activity ◽  
Coagulation Factors ◽  
Clotting Time ◽  
Inhibitory Antibody ◽  
Clot Strength ◽  
Low Concentration ◽  
Extrinsic Cues ◽  
Coagulation And Fibrinolysis

Abstract Abstract 2256 Blood coagulation is marked by sequential proteolytic activation of the coagulation factors and complex feedback modulation which is central to haemostasis. The fine balance between the coagulation cascade and the fibrinolytic system is also an integral component in haemostasis. Multiple coagulation factors, including FIIa (thrombin), FXa, FXIa, and FXIIa, are attractive targets for treating thrombosis, and novel inhibitors for several targets are already in late stage development or on the market. However, the responsiveness of these inhibitors to intrinsic vs. extrinsic cues warrants further study because in different disease settings the relative signaling intensities from the two pathways could be different in vivo. It is also poorly understood how these inhibitors might differentially modulate fibrinolytic activity, and a dysregulated fibrinolytic cascade is a potential contributing factor to both thrombosis and bleeding. We therefore sought to gain insight by comparing effects of four agents (dabigatran, apixaban, FXIa inhibitory antibody (FXIa Ab), and FXIIa inhibitory antibody (FXIIa Ab)) in whole blood coagulation and fibrinolysis in vitro using thromboelastography (TEG). We first established optimal clot-triggering conditions for TEG by using either a low concentration of kaolin (approximately 100-fold less kaolin than in standard aPTT assay), or a low concentration of tissue factor (TF) (1:8000 dilution of Recombiplastin in blood), along with recalcification. We then performed titration of tissue plasminogen-activator (tPA) with these clotting conditions and identified a tPA concentration of 125 ng/ml as optimal in inducing a fibrinolytic phase without significantly altering the clotting phase. Compound profiling was then pursued by dose titration with the individual agent in normal human donor's blood and tested TEG responses to kaolin+/− tPA and TF+/−tPA. Key TEG parameters that were assessed include R (clotting time), reflecting anticoagulant effect, MA (maximal amplitude), representing clot strength, and Ly30 (percent lysis at 30 minutes after MA), reflecting fibrinolytic activity. For clotting time, dabigatran and apixaban both delayed R in response to both kaolin and TF. FXIa Ab and FXIIa Ab delayed R only in response to kaolin and not to TF. This is consistent with the understanding that thrombin and FXa are in the common pathway, and FXIa and FXIIa are in the intrinsic pathway. For agents that affected the response to kaolin, stack-ranking of the magnitude of effect was FXIa Ab > dabigatran > apixaban > FXIIa Ab. For agents that affected the response to TF, dabigatran was slightly more effective than apixaban. Regarding clot strength, none of the agents appreciably altered MA in the absence of tPA; in the presence of tPA, dabigatran, apixaban, and FXIa Ab reduced MA to a greater extent than FXIIa Ab. Regarding fibrinolytic activity, none of the agents modulated Ly30 in the absence of tPA, consistent with the notion that these inhibitors alone are not fibrinolytic agents. However, in the presence of tPA, dabigatran, apixaban, FXIa Ab, but not FXIIa Ab, enhanced Ly30, suggesting that inhibition of thrombin, FXa, or FXIa may potentiate tPA-induced fibrinolysis. In summary, each inhibitory agent examined with this whole blood spike-in paradigm exhibited a distinct profile of effects on clotting time, clot strength and fibrinolysis with intrinsic and extrinsic cues in the presence and absence of tPA. Because the thrombotic cues in different patient segments are highly complex, and the endogenous fibrinolytic activities in different tissues may vary considerably, our results provide a benchmarking dataset for these inhibitory agents and their respective targets and represent a step forward in differentiating specific agents and targets in our effort to optimize safety and efficacy with novel anticoagulant therapies. Disclosures: Chen: Merck Research Laboratories: Employment. Wu:Merck Research Laboratories: Employment. Xu:Merck Research Laboratories: Employment. Ogletree:Merck Research Laboratories: Employment. Plump:Merck Research Laboratories: Employment. Graziano:Merck Research Laboratories: Employment. Wang:Merck Research Laboratories: Employment.

scholarly journals Role of Serotonin in Blood Coagulation

1957 ◽  
Vol 189 (3) ◽  
pp. 470-474 ◽  
Author(s):  
W. L. Milne ◽  
S. H. Cohn
Keyword(s):  
Blood Coagulation ◽  
Therapeutic Effect ◽  
Whole Blood ◽  
Active Role ◽  
Clotting Time ◽  
Platelet Factor ◽  
Coagulation Defect ◽  
Increased Sensitivity

Effects of serotonin (5HTA) were studied a) in whole blood ( in vitro) from both normal and x-irradiated animal, and b) in isolated clotting systems of plasma and purified fibrinogen. 5HTA was found to play an active role in blood coagulation in addition to its previously demonstrated role as a vasoconstrictor. In whole blood from normal and irradiated animals, and in platelet-deficient plasma, 5HTA acts in a manner similar to platelet factor 2 in accelerating the conversion of fibrinogen to fibrin. In a purified fibrinogen solution, 5HTA inhibits the fibrinogen-fibrin reaction, and thus differs from platelet factor 2. This may be due to the absence of an antithrombin in the purified fibrinogen solution. Serotonin appears to have a therapeutic effect on the postirradiation coagulation defect in that it decreases the prolonged heparin clotting time. This increased sensitivity to heparin during the postirradiation thrombocytopenia may be due to a deficiency of serotonin, which acts as an antagonist to heparin (an antithrombin). The action of 5HTA is postulated as an antagonist of an antithrombin which blocks the fibrinogen to fibrin reaction.

Antiplatelet agents in tissue factor–induced blood coagulation

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2314-2322 ◽  
Author(s):  
Saulius Butenas ◽  
Kevin M. Cawthern ◽  
Cornelis van't Veer ◽  
Maria E. DiLorenzo ◽  
Jennifer B. Lock ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Antiplatelet Agents ◽  
Receptor Activation ◽  
Clotting Time ◽  
Ligand Interaction ◽  
Clot Time

Abstract Several platelet inhibitors were examined in a tissue factor (TF)–initiated model of whole blood coagulation. In vitro coagulation of human blood from normal donors was initiated by 25 pM TF while contact pathway coagulation was suppressed using corn trypsin inhibitor. Products of the reaction were analyzed by immunoassay. Preactivation of platelets with the thrombin receptor activation peptide did not influence significantly the clotting time or thrombin–antithrombin III complex (TAT) formation. Addition of prostaglandin E1 (5 μM) caused a significant delay in clotting (10.0 minutes) versus control (4.3 minutes). The prolonged clotting time is correlated with delays in platelet activation, formation of TAT, and fibrinopeptide A (FPA) release. In blood from subjects receiving acetylsalicylic acid (ASA or aspirin), none of the measured products of coagulation were significantly affected. Similarly, no significant effect was observed when 5 μM dipyridamole (Persantine) was added to the blood. Antagonists of the platelet integrin receptor glycoprotein (gp) IIb/IIIa had intermediate effects on the reaction. A 1- to 2-minute delay in clot time and FPA formation was observed with addition of the antibodies 7E3 and Reopro (abciximab) (10 μg/mL), accompanied by a 40% to 70% reduction in the maximal rate of TAT formation and delay in platelet activation. The cyclic heptapetide, Integrilin (eptifibatide), at 5 μM concentration slightly prolonged clot time and significantly attenuated the maximum rate of TAT formation. The disruption of the gpIIb/IIIa-ligand interaction not only affects platelet aggregation, but also decreases the rate of TF-initiated thrombin generation in whole blood, demonstrating a potent antithrombotic effect superimposed on the antiaggregation characteristics.

Evidence That Fibrinogen Inhibits Leukocyte Adhesion to Fibrin Clot and Immobilized Fibrinogen by Binding to the Substrate but Not to Integrins.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2631-2631
Author(s):  
Valeryi K. Lishko ◽  
Tatiana P. Ugarova
Keyword(s):  
Leukocyte Adhesion ◽  
Fibrin Clot ◽  
Plasma Fibrinogen ◽  
Flow Conditions ◽  
Fibrin Gel ◽  
Surface Receptors ◽  
High Concentrations ◽  
Adhesive Interactions ◽  
Adhesive Effect

Abstract The recruitment of phagocytic leukocytes to sites of injured vessel wall plays an important role in thrombus remodeling during normal vascular repair and in the pathophysiology of thrombosis. Fibrin and fibrinogen, present in the thrombus, are potent adhesive substrates for neutrophils and monocytes. They support cellular attachment by binding cell surface receptors that belong to the β2 subfamily of integrins. Adhesive interactions of neutrophils and monocytes with polymerized fibrin and insoluble fibrinogen matrix in vivo occur in the presence of high concentrations of circulating plasma fibrinogen (~2–4 mg/ml). One important property of fibrinogen that would have a major bearing on leukocyte adhesion is its capacity to form complexes with fibrin. Therefore, by virtue of its binding to the fibrin clot and/or immobilized fibrinogen, soluble plasma fibrinogen can influence leukocyte adhesion to these substrates. In this study, the possibility that soluble fibrinogen could protect fibrin from adhesion of leukocytes was examined. Fibrinogen was an efficient inhibitor of adhesion of U937 monocytoid cells and neutrophils to fibrin gel and immobilized fibrin(ogen). An investigation of the mechanism by which fibrinogen exerts its influence on leukocyte adhesion indicated that it did not block integrins but rather associated non-covalently and weakly with fibrin(ogen) substrates. Consequently, leukocyte integrins that engage fibrinogen molecules loosely bound to the fibrin(ogen) matrix are not able to consolidate their grip on the substrate; subsequently, cells detach. This conclusion is based on the evidence obtained in adhesion studies using various β2-integrin bearing cells and performed under static and flow conditions. Furthermore, surface plasmon resonance studies, undertaken to determine the Kd of fibrinogen-fibrin interactions under flow conditions, indicated that fibrinogen formed complexes with fibrin(ogen) with micromolar affinities. Thus, these findings reveal a new role of fibrinogen in integrin-mediated leukocyte adhesion. They also imply that the anti-adhesive effect of fibrinogen may protect the thrombus from an excessive leukocyte accumulation and premature dissolution at the early stages of wound healing when hemostatic plug integrity is critical for preventing blood loss.

scholarly journals FTIR Investigation of Nitrogen Monoxide and Carbon Monoxide Influence on Human Blood Coagulation

2021 ◽  
Vol 12 (5) ◽  
pp. 7102-7110
Keyword(s):  
Blood Plasma ◽  
Blood Coagulation ◽  
Prolonged Exposure ◽  
Nitrogen Monoxide ◽  
Fibrin Clot ◽  
Hydrate Shell ◽  
Blood Plasma Sample ◽  
High Concentrations ◽  
The One ◽  
Α Helix

The formation of fibrin clots determines the characteristics of blood plasma coagulation. It is known that small molecules like nitrogen and carbon monoxides have a toxic effect at high concentrations due to the competitive binding to hemoglobin, preventing further oxygen transfer. The mechanisms of intoxication with these gases have been extensively studied in the literature, but there is little information on their effects on other vital processes. In particular, blood coagulation parameters have not been studied, although prolonged exposure to relatively low concentrations of gases can cause significant pathological changes. In this paper, the characteristics of the fibrin-polymer after coagulation of a blood plasma sample under conditions of pretreatment with gases were studied using FTIR-spectroscopy. The changes in the vibrations of individual bonds and fragments in the Amide I and Amide B regions are shown and analyzed. It was established that at concentrations of CO exceeding the endogenous levels, the connector α-helix unfolds and β-structures form, leading to the loss of part of the hydrate shell, the formation of a fibrin clot with a disordered structure of higher density due to an increase in its hydrophobicity. For cases where samples were treated with NO gas, the degree of aggregation of the fibrin clot from plasma incubated with nitroglycerin was one-quarter less than the original, the proportion of α-helices was not reduced, and there were no disordered structures in the clot. This may indicate a lower clot density and probably easier lysis of the one.

Effect of acidity on blood coagulation

1961 ◽  
Vol 201 (2) ◽  
pp. 379-382 ◽  
Author(s):  
Jack W. Crowell ◽  
Bobby Houston
Keyword(s):  
Lactic Acid ◽  
Blood Coagulation ◽  
Whole Blood ◽  
Fast Moving ◽  
Circulatory Arrest ◽  
Clotting Time ◽  
Acid Base ◽  
Acid Injection ◽  
Dog Blood

Various methods were used to determine the effect of decreasing the pH on the coagulation of whole blood. It was found that injection of lactic acid into the slowly moving blood of an A-V shunt caused death in dogs much faster than the same rate of acid injection into the fast-moving blood of the aorta. Also, it was found that lowering the pH of withdrawn heparinized blood made it toxic upon reinjection, whereas blood withdrawn and not treated, treated with the neutralized acid-base mixture, or to which a larger amount of heparin had been added was not toxic upon reinjection. Heparinized blood withdrawn from a dog and to which various amounts of lactic acid were added showed a decrease in its clotting time as the pH became lower and lower. Below a pH of approximately 6.7, the heparinized blood coagulated almost as rapidly as nonheparinized blood. Finally, it was found that there was clotting in ligated vascular tubes of dogs after lowering the pH of the blood, whereas no clotting occurred in vascular tubes ligated before the acid was given or after the pH of the dog blood had returned to normal. Emphasis is placed on the fact that the decrease in pH in dogs subjected to circulatory arrest is associated with intravascular clotting.

Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

1992 ◽  
Vol 68 (06) ◽  
pp. 694-700 ◽  
Author(s):  
Roy R Hantgan ◽  
Silvia C Endenburg ◽  
I Cavero ◽  
Gérard Marguerie ◽  
André Uzan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Integrin Receptor ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Receptor Recognition ◽  
Adhesive Interactions ◽  
Coated Surfaces ◽  
Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

Effect of Temperature, Velocity Gradient and I.V. Heparin on in Vitro Blood Coagulation and Platelet Aggregation

1967 ◽  
Vol 17 (01/02) ◽  
pp. 112-119 ◽  
Author(s):  
L Dintenfass ◽  
M. C Rozenberg
Keyword(s):  
Blood Coagulation ◽  
Velocity Gradient ◽  
Elevated Temperatures ◽  
Morphological Changes ◽  
Effect Of Temperature ◽  
Clotting Time ◽  
Progressive Change ◽  
Aggregation Of Platelets ◽  
Microscopic Techniques

SummaryA study of blood coagulation was carried out by observing changes in the blood viscosity of blood coagulating in the cone-in-cone viscometer. The clots were investigated by microscopic techniques.Immediately after blood is obtained by venepuncture, viscosity of blood remains constant for a certain “latent” period. The duration of this period depends not only on the intrinsic properties of the blood sample, but also on temperature and rate of shear used during blood storage. An increase of temperature decreases the clotting time ; also, an increase in the rate of shear decreases the clotting time.It is confirmed that morphological changes take place in blood coagula as a function of the velocity gradient at which such coagulation takes place. There is a progressive change from the red clot to white thrombus as the rates of shear increase. Aggregation of platelets increases as the rate of shear increases.This pattern is maintained with changes of temperature, although aggregation of platelets appears to be increased at elevated temperatures.Intravenously added heparin affects the clotting time and the aggregation of platelets in in vitro coagulation.

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