In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

2010 ◽  
Vol 104 (09) ◽  
pp. 514-522 ◽  
Author(s):  
Thomas Lecompte ◽  
Agnès Tournier ◽  
Lise Morlon ◽  
Monique Marchand-Arvier ◽  
Claude Vigneron ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Cathepsin G ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Factor V ◽  
Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

Microfluidic Tools To Probe the Spatial Dynamics of Coagulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3934-3934
Author(s):  
Christian J. Kastrup ◽  
Matthew K. Runyon ◽  
Feng Shen ◽  
Rustem F. Ismagilov
Keyword(s):  
Surface Chemistry ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Microfluidic Device ◽  
Spatial Dynamics ◽  
Vascular Damage ◽  
Coagulation Cascade ◽  
Clotting Factors ◽  
Micrometer Scale

Abstract To investigate the biophysical mechanisms that regulate the spatial dynamics of blood coagulation, we have developed a set of microfluidic tools that allow analysis and perturbation of blood coagulation on the micrometer scale with precise control of fluid flow, geometry, and surface chemistry. Physiological coagulation occurs in a localized manner; specifically, coagulation is believed to occur exclusively at regions of substantial vascular damage and does not spread throughout the entire vascular system. In vitro analysis and characterization of these spatial dynamics requires the ability to reproduce and perturb this system, an ability that is not provided by the mixed reactor systems commonly used for in vitro studies of blood coagulation. We developed microfluidic devices with micrometer-scale channels and methods to coat these channels with various phospholipids, including components of the blood coagulation network such as thrombomodulin and tissue factor, to reproduce in vitro the geometry and surface chemistry of blood vessels in vitro. In a microfluidic device with channels coated with phospholipids and thrombomodulin, we demonstrated that clots propagate in a wave-like fashion with a constant velocity in the absence of flow. We also showed that propagation of coagulation from an occluded channel to a channel with flowing blood plasma can be regulated by the geometry of the junction and the shear rate in the channel with flowing plasma. We also developed microfluidic tools to probe the spatial dynamics of initiation of clotting by patterning surfaces with tissue factor reconstituted into phospholipids bilayers. When human plasma or whole blood was exposed to these surfaces in a microfluidic device, clotting occurred only on patches of tissue factor larger than a threshold size. This threshold patch size is controlled by the rate of activation of clotting factors at the patch and the rate of transport of activated factors off the patch. These results suggest a mechanism for how tissue factor can circulate in blood without causing clotting, and how small regions of vascular damage can exist without causing clotting. These results also suggest new biophysical mechanisms that may control interactions between the coagulation cascade and bacterial surfaces.

scholarly journals Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram

2009 ◽  
Vol 102 (11) ◽  
pp. 936-944 ◽  
Author(s):  
Kellie Machlus ◽  
Emily Colby ◽  
Jogin Wu ◽  
Gary Koch ◽  
Nigel Key ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Relative Sensitivity ◽  
Epidemiological Studies ◽  
Peak Height ◽  
Clotting Factor ◽  
Time To Peak ◽  
High Throughput Method ◽  
Assay Conditions

SummaryElevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels.

scholarly journals High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

2021 ◽  
Vol 22 (18) ◽  
pp. 9705
Author(s):  
Sara Bernal ◽  
Irene Pelaez ◽  
Laura Alias ◽  
Manel Baena ◽  
Juan A. De Pablo-Moreno ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Coagulation Disorder ◽  
Factor V ◽  
Factor V Deficiency ◽  
Advanced Therapies ◽  
Factor V Gene ◽  
V Gene

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

scholarly journals An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3067-3072 ◽  
Author(s):  
Cornelis van ‘t Veer ◽  
Neal J. Golden ◽  
Michael Kalafatis ◽  
Paolo Simioni ◽  
Rogier M. Bertina ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor V ◽  
Severe Hemophilia ◽  
Factor Viii Activity ◽  
Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

Differential Effects of TAK-442, a Factor Xa Inhibitor, and Ximelagatran, a Thrombin Inhibitor, on Bleeding: Possible Role of Differences in Effects on Factor V-Mediated Feedback on Blood Coagulation Cascade

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5460-5460
Author(s):  
Noriko Konishi ◽  
Katsuhiko Hiroe ◽  
Yasuhiro Imaeda ◽  
Takuya Fujimoto ◽  
Keiji Kubo ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Rat Model ◽  
Positive Feedback ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Factor V ◽  
Differential Effects

Abstract Thrombin generation serves to amplify the coagulation cascade via positive feedback activation of factor V (FV) and factor VIII. We hypothesized that factor Xa (FXa) inhibitors, unlike thrombin inhibitors, would not block the feedback activation of the coagulation cascade but would have a favorable anticoagulating profile—sufficient to prevent thrombus formation, yet not interfere with hemostatic plug formation. TAK-442 is a newly synthesized, selective FXa inhibitor that strongly inhibits FXa (with a Ki value of 1.8 nM), and displays more than 440x selectivity toward FXa than other serine proteases. In the present study, we compared the effects of TAK-442 versus ximelagatran on FV-mediated positive feedback in vitro, and on their antithrombotic and hemorrhagic effects in a rat model of venous thrombosis. In vitro, TAK-442 gradually inhibited thrombin generation and prolonged prothrombin time (PT) in a dose-dependent manner, while melagatran, an active form of ximelagatran, exhibited a steeper effect at higher doses tested. The PT prolonging potency was increased in FV–deficient human plasma, with CT2 values (the concentration that causes 2 times prolongation of clotting times) of 120 nM for TAK-442 and 32 nM for melagatran, compared with 500 nM and 360 nM for TAK-442 and melagatran, respectively in normal plasma. In the rat model of venous thrombosis, TAK-442 (10 mg/kg, po) prevented thrombus formation by 55% and prolonged PT by 1.3 times of control values; a similar effect was observed in ximelagatran-treated (3 mg/kg, po) animals, with 59% inhibition of thrombus formation and 1.2 times prolongation of PT. TAK-442 at 100 mg/kg, prolonged PT by 2.1 times, with no significant change in bleeding time (BT); in contrast, increasing the dose of ximelagatran to 10 mg/kg, po prolonged PT by 3.9 times and significantly (P&lt;0.025) increased BT. Our data suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin

2006 ◽  
Vol 96 (09) ◽  
pp. 317-324 ◽  
Author(s):  
Chenliang Wu ◽  
Jon Vincelette ◽  
Baby Martin-McNulty ◽  
Serene Alexander ◽  
Brent Larsen ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Neutralizing Antibodies ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Coagulation Cascade ◽  
Control Group ◽  
Therapeutic Effects ◽  
Single Chain ◽  
Single Chain Antibody

SummaryTissue factor (TF) exposure isa potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TF-neutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibits TF/factor VIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. Ina rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in ∼60% of the animals and a clinical score of ∼2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neither Ab(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED50, nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10– to 100-fold enhancement of the anticoagulant potency, relative to the ED50 in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.

scholarly journals The Blood Clotting Properties of Rabbit Peritoneal Leukocytes in Vitro

1967 ◽  
Vol 17 (01/02) ◽  
pp. 222-236 ◽  
Author(s):  
S. I Rapaport ◽  
P. F Hjort
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
In Vitro Study ◽  
Factor V ◽  
Clotting Factors ◽  
Clotting System ◽  
Procoagulant Effect ◽  
Peritoneal Leukocytes ◽  
Thromboplastic Activity

SummaryA systematic in vitro study has been carried out of the blood clotting properties of rabbit peritoneal leukocytes. Rabbit heterophils have been shown to possess weak but definite tissue thromboplastic activity. They also contain an anticoagulant activity which is active in the intrinsic clotting system, in the extrinsic clotting system with brain thromboplastin, and in clotting systems with Russell’s viper venom. When the thromboplastic activity of the white cell itself initiates clotting, the anticoagulant is much less effective and the procoagulant effect of the WBC suspension predominates. Rabbit heterophils do not bind plasma clotting factors, do not activative factor V, and, under our conditions, do not precipitate fibrinogen from normal plasma, from plasma exposed to traces of thrombin in vitro, or from plasma of rabbits given endotoxin.The relation of these findings to the role of the granulocyte in the pathogenesis of the generalized Shwartzman reaction has been discussed.

The Resusciatative Fluid You Chose May Potentiate Bleeding.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1029-1029
Author(s):  
Kathleen E. Brummel-Ziedins ◽  
Matthew F. Whelihan ◽  
Eduards G. Ziedins ◽  
Kenneth G. Mann
Keyword(s):  
Hemorrhagic Shock ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
In Vitro Study ◽  
The United States ◽  
Fibrin Clot ◽  
Blood Dilution ◽  
Lactated Ringer's Solution

Abstract Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation to be used (dilutions ranging up to 66%) in hemorrhagic shock for restoring hemodynamic stability. Trauma results in massive exposure of tissue factor to the circulation and explosive amounts of thrombin being generated. We studied the effect of various resuscitative formulas to blood coagulation, specifically thrombin generation and fibrin formation, in a controlled setting using corn trypsin inhibited whole blood initiated with a 5pM stimulus of tissue factor. Thrombin generation measured as its complex with antithrombin III (TAT) was evaluated periodically over a time course of 20 min. Fibrin clots were collected and weighed. We investigated four diluents (0.9% NaCl (NS), lactated Ringer’s solution (LR), 6% hydroxyethyl starch (HES) and 3% NaCl (HS)) each at a 0,10, 20, 30, 40, 50 and 60% blood dilution. At a 10% dilution TAT generation was in the order of LR (−4%) &lt; HES (−8%)&lt; NS and HS (−12%). Diluting by 20% resulted in further decreases of TAT formation. The fibrin clot mass decreased dramatically with a 20% dilution for NS (−42%) and HES (−30%). Conversely, HS produced no change in fibrin mass but effected the largest change in thrombin generation rate (−56%). At a 50% dilution, comparable thrombin generation profiles were obtained for LR, HES and NS (~35% decrease). However, the fibrin masses decreased by 27% with LR, 46% with NS and 74% with HES. No clot formation or thrombin generation was seen with HS at &gt; 20% dilution. This in vitro study shows that: 1) LR has the least effect on thrombin generation and gave higher than anticipated clot weights; 2) HES reduced the fibrin clot mass at higher dilutions; 3) HS abolishes coagulation after a 20% dilution. Overall, both the extent and nature of hemodilution cause profound alterations in the hemostatic mechanism.

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