Thrombin generation and fibrin clot formation under hypothermic conditions: An in vitro evaluation of tissue factor initiated whole blood coagulation

SummaryBlood dilution after transfusion fluids leads to diminished coagulant activity monitored by rotational thromboelastometry, assessing elastic fibrin clot formation, or by thrombin generation testing. We aimed to determine the contributions of blood cells (platelets, red blood cells) and plasma factors (fibrinogen, prothrombin complex concentrate) to fibrin clot formation under conditions of haemodilution in vitro or in vivo. Whole blood or plasma diluted in vitro was supplemented with platelets, red cells, fibrinogen or prothrombin complex concentrate (PCC). Thromboelastometry was measured in whole blood as well as plasma; thrombin generation was determined in parallel. Similar tests were performed with blood from 48 patients, obtained before and after massive fluid infusion during cardiothoracic surgery. Addition of platelets or fibrinogen, in additive and independent ways, reversed the impaired fibrin clot formation (thromboelastometry) in diluted whole blood. In contrast, supplementation of red blood cells or prothrombin complex concentrate was ineffective. Platelets and fibrinogen independently restored clot formation in diluted plasma, resulting in thromboelastometry curves approaching those in whole blood. In whole blood from patients undergoing dilution during surgery, elastic clot formation was determined by both the platelet count and the fibrinogen level. Thrombin generation in diluted (patient) plasma was not changed by fibrinogen, but improved markedly by prothrombin complex concentrate. In conclusion, in dilutional coagulopathy, platelets and fibrinogen, but not red blood cells or vitamin K-dependent coagulation factors, independently determine thromboelastometry parameters measured in whole blood and plasma. Clinical decisions for transfusion based on thromboelastometry should take into account the platelet concentration.

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Demystifying Tissue Factor.

Blood ◽

10.1182/blood.v104.11.1936.1936 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1936-1936 ◽

Cited By ~ 2

Author(s):

Saulius Butenas ◽

Matthew T. Gissel ◽

Beth A. Bouchard ◽

Kathleen E. Brummel ◽

Behnaz Parhami-Seren ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Whole Blood ◽

Thrombin Generation ◽

Factor Viia ◽

Integral Membrane Protein ◽

Factor X ◽

Initiation Phase

Abstract Tissue factor (TF) is an integral membrane protein, which is the key initiator of blood coagulation in vivo. Due to the limited availability of natural TF, recombinant proteins of various lengths and origins have been extensively used in research and clinical laboratories worldwide. Experimental results acquired with recombinant TF proteins are frequently used for the understanding of the coagulation processes occurring in vivo, although there is a lack of data confirming the structural and functional identity of natural TF proteins from various sources and recombinant ones. In the current study, human TF from cultured monocytes and purified from placenta were compared with three different species of recombinant TF: 1–218 (extracellular domain only), 1–242 (lacking cytoplasmic domain) and 1–263 (full-length). Anti-TF mAbs gave 93–98% inhibition of TF activity for all TF species tested, in both natural and relipidated preparations. It was established that purified placental TF has a higher affinity for factor VIIa (Kd 0.13 nM) than recombinant counterparts 1–242 and 1–263 (Kd 0.50–0.80 nM). Similarly, placental TF is more efficient in factor X activation by the extrinsic Xase than recombinant TF 1–242 (the second order rate constants are 3.0x107 and 0.7x107 M−1s−1, respectively). We explored the use of these TF species as well as monocyte TF (purified/relipidated and present on LPS-stimulated monocytes) for the initiation of thrombin generation in two in vitro models of blood coagulation. At equimolar concentrations (5 pM; determined by immunoassay), when evaluated in synthetic plasma reconstituted with 2x108/ml platelets, recombinant TF 1-263 provided an initiation phase of ~4 min. Placental TF and relipidated monocyte TF had similar profiles of thrombin generation with an initiation phase of ~3 min. In contrast, 0.5 pM TF on LPS-stimulated monocytes gave an initiation phase of ~1 min. Even at 0.05 pM concentration, monocyte TF was as active as any relipidated protein at 5.0 pM. A similar pattern of relative TF activity was observed in whole blood and plasma PT clotting assays. TF on stimulated monocytes gave the highest activity, exceeding that of any relipidated protein by 100–200-fold. Recombinant TF 1–242 was more active than recombinant TF 1–263 and placental TF in the PT assay but less active in synthetic plasma and whole blood. The lowest overall activity was observed for relipidated monocyte TF. Our data suggest that TF proteins from different sources are different with respect to their functional properties.

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Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽

10.1182/blood.v112.11.3736.3736 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3736-3736

Author(s):

Anna Falanga ◽

Alfonso Vignoli ◽

Marina Marchetti ◽

Laura Russo ◽

Marina Panova-Noeva ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Thrombin Generation ◽

Procoagulant Activity ◽

Jak2v617f Mutation ◽

Wild Type ◽

Coagulation Activation ◽

Allele Burden ◽

Generation Capacity

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

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Antiplatelet agents in tissue factor–induced blood coagulation

Blood ◽

10.1182/blood.v97.8.2314 ◽

2001 ◽

Vol 97 (8) ◽

pp. 2314-2322 ◽

Cited By ~ 62

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.

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Bovine Heparin Compared to Porcine Heparin to Demonstrate Bioequivalence

Blood ◽

10.1182/blood-2018-99-118809 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1253-1253

Author(s):

Marco Khiella ◽

Walter Jeske ◽

Jeanine Walenga ◽

Omer Iqbal ◽

Rajan Laddu ◽

...

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Maximum Amplitude ◽

Clot Formation ◽

K Value ◽

Significant Difference ◽

Heparin Monitoring ◽

The U.S

Abstract Introduction: Heparin prevents blood clots from forming in patients undergoing heart surgeries, dialysis, multiple other procedures, and for medical treatment of thrombosis such as associated with cancer. Currently, all heparin products in the U.S. are derived from the intestinal mucosa of pigs. Seventy-five percent of the crude porcine heparin used to make the active pharmaceutical ingredient (API) comes from outside the U.S., with a majority originating from China. Reintroduction of bovine heparin into the U.S. market would expand sources for this critical drug, thus addressing concerns about potential shortages and product adulteration. This study was undertaken to determine the bioequivalence of bovine and porcine heparins using assays relevant to the clinical setting. The assays used in this study were chosen because they overcome limitations of the conventional APTT for heparin monitoring. Selected TEG and ACT assays use whole blood which better simulates physiologic conditions and assesses the full in vitro anticoagulation potential of heparin; these assays are also routinely used clinically for heparin monitoring. The thrombin generation assay is state-of-the-art for hemostatic clinical lab testing, and it provides a more sensitive endpoint of the final generation of thrombin once coagulation is activated. Heparins were studied at concentrations used clinically and tested by assays sensitive to these concentrations. Methods: Bovine heparin API (BH; 7 lots) and US clinical grade porcine heparin (PH: 3 lots) were tested in parallel using recalcified whole blood thromboelastography (TEG; Haemonetics), celite activated clotting time (ACT; Hemochron), and thrombin generation (TGA; Diapharma). Fresh blood was obtained from healthy volunteers under an IRB approved protocol (n=6 per group). Previous work from our lab (Jeske W, Thrombosis & Hemostasis Societies of North America, P57, 2018) identified a weaker potency of BH than PH when compared on an equigravimetric basis in pharmacopeial assays; however, equivalent potency could be obtained when BH was standardized against PH on a unitage basis. In this study, heparins were studied on both a gravimetric and a unitage basis for a comprehensive evaluation. The potency of the BH was determined by pharmacopeial compliant anti-Xa and anti-IIa chromogenic assays cross-referenced to the porcine USP Heparin Reference Standard. Results: All results are depicted in Table 1. For the TEG, the R value, time of latency from start to initial fibrin formation, and the K value, time to achieve a defined clot strength due to thrombin generation and platelet activation, bovine heparin and porcine heparin did not demonstrate a significant difference in anticoagulant activity. The TEG angle, a measure of the speed of fibrin build-up and cross-linking (clot strengthening or rate of clot formation), and the maximum amplitude (MA) value, a function of the maximum of fibrin and platelet binding representing the strongest point of fibrin clot formation, also revealed no significant difference between bovine and porcine heparin anticoagulant activity. For the ACT, at concentrations of 10 µg/mL and 25 µg/mL, there were no statistically significant differences between BH and PH. For the TGA, measuring the time delay until the initiation of thrombin generation following tissue factor (TF) activation, the highest amount of thrombin generated after TF activation, and the total amount of thrombin generated after TF activation (AUC), showed a trend that PH was more potent than BH, but wide variation in the results did not allow for statistical differences to be identified. Conclusion: The results of this investigation demonstrate that bovine heparin produces an equivalent anticoagulant activity as porcine heparin in the TEG, ACT, and TGA assay systems. The equivalent ACT results, in particular, were unexpected since gravimetric amounts of heparins were evaluated. As for all heparins, standardization of bovine heparin in accordance with the process used for porcine heparin will assure equivalent anticoagulant activity among bovine and porcine heparins in whole blood, plasma-based, and pharmacopeial assays. This study further demonstrates that the current assays used to monitor porcine heparin can be similarly used to monitor bovine heparin. Disclosures Walenga: Eurofarma: Research Funding.

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A Variant of Recombinant Factor VIIa with Enhanced Procoagulant and Antifibrinolytic Activities in a Model of Hemophilia.

Blood ◽

10.1182/blood.v108.11.767.767 ◽

2006 ◽

Vol 108 (11) ◽

pp. 767-767 ◽

Cited By ~ 1

Author(s):

Alisa S. Wolberg ◽

Egon Persson ◽

Ulla Hedner ◽

Mirella Ezban ◽

Geoffrey A. Allen

Keyword(s):

Thrombin Generation ◽

Factor Viia ◽

Factor Xa ◽

Fibrin Clot ◽

Clot Formation ◽

Factor X ◽

High Concentrations ◽

Vitro Model ◽

Factor X Activation

Abstract Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. We have shown that high concentrations of rFVIIa improve parameters of thrombin generation and fibrin clot formation and stability in an in vitro model of hemophilia (Wolberg et al. 2005 Brit J Haem 131:645–55). Recently, new rFVIIa analogs with increased procoagulant activity have been described. One of these analogs, rFVIIa with mutations V158D/E296V/M298Q (NN1731), exhibits increased activity in in vitro and in vivo models. In the present study, we compared the abilities of rFVIIa and NN1731 to specifically modulate factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability in an in vitro model of hemophilia. RFVIIa and NN1731 similarly increased factor X activation on tissue factor−bearing monocytes; however, NN1731 exhibited 30−fold higher factor Xa generation rates on activated platelets than similar concentrations of rFVIIa. We employed a complete, cell−based, reconstituted model of coagulation to compare the activities of rFVIIa and NN1731 in hemophilic conditions. The model system consists of tissue−factor bearing monocytes, purified pro− and anti−coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), and freshly−isolated, unactivated platelets. Fibrinogen is included in certain wells to examine clot formation. “Hemophilia” is simulated by omitting factors VIII and IX. In this assay, NN1731 produced 4 – 10−fold higher maximal thrombin generation rates than equal concentrations of rFVIIa. In contrast to even very high concentrations of rFVIIa (up to 500 nM), NN1731 was able to normalize the rate of thrombin generation. NN1731 did not directly activate platelets and thrombin generation was not detected prior to platelet activation, suggesting that the presence of NN1731 would not lead to thrombin generation in the absence of injury. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at lower concentrations than were required of rFVIIa. In a plasmin challenge assay, in which clot formation is initiated in the presence of plasmin, both rFVIIa and NN1731 increased fibrin formation and the length of time that fibrin was present in the absence of factors IX and VIII; however, NN1731 was effective at lower concentrations than were required of rFVIIa. Our results indicate that NN1731 increases factor Xa and thrombin generation and promotes clot formation and stability to a greater degree than equal concentrations of rFVIIa. For these reasons, NN1731 may be especially efficacious in situations in which rapid formation of a firm fibrin clot with increased resistance to fibrinolysis is necessary to achieve hemostasis.

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Idarucizumab Fully Restores Dabigatran-Induced Alterations on Platelet and Fibrin Deposition on Damaged Vessels: Studies in Vitro with Circulating Human Blood

Blood ◽

10.1182/blood.v124.21.2878.2878 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2878-2878 ◽

Cited By ~ 8

Author(s):

Eduardo Arellano-Rodrigo ◽

Irene Lopez-Vilchez ◽

Patricia Molina ◽

Marcos Pino ◽

Maribel Diaz-Ricart ◽

...

Keyword(s):

Human Blood ◽

Whole Blood ◽

Thrombin Generation ◽

Clot Formation ◽

Fibrin Deposition ◽

Flow Conditions ◽

Emergency Procedures ◽

Platelet Deposition ◽

Baseline Values

Abstract BACKGROUND: Despite the proven efficacy and safety profile of dabigatran as compared to warfarin, bleeding remains a concern as with all anticoagulants and the reversal of dabigatran’s anticoagulant effect for emergency procedures remains controversial. Recently, idarucizumab, a specific antidote for dabigatran, has been functionally characterized and its efficacy demonstrated in animal models and healthy volunteer studies. AIMS: We explored the effects of dabigatran on hemostasis in human blood focusing on possible interference with platelet and coagulation responses to vessel injury under flow conditions. We also compared the potential efficacy of idarucizumab with procoagulant strategies such as prothrombin complex concentrates (PCC), activated PCC (aPCC) or rFVIIa at reversing the antithrombotic action of dabigatran to better understand local processes in response to injury. METHODS: Concentrations of dabigatran equivalent to the Cmax reported at steady state after therapy with 150 mg twice daily (184 ng/mL) were added in vitro to blood aliquots from 11 healthy donors. Whole blood samples were used to evaluate modifications in different coagulation biomarkers: 1) fibrin and platelet deposition on damaged vascular segments with whole blood under flow conditions at a shear rate of 600 s-1, 2) dynamics of thrombin generation (TG) in plasma using a fluorogenic assay (Technothrombin TGA) and 3) viscoelastic parameters of clot formation in whole blood using by thromboelastometry (ROTEM) The efficacy of specific reversal with idarucizumab 0.3, 1 and 3 mg/mL was compared with that of non specific procoagulant concentrates such as aPCC 25 and 75 IU/kg, PCC 70 IU/kg, or rFVIIa 120 µg/kg. RESULTS: Dabigatran (184 ng/mL) caused a pronounced 85% reduction of fibrin coverage on the damaged vessel from 67.2±9.8 to 9.5±1.3 % (p<0.01) and a moderate 35% reduction of platelet deposition from 25.9±2.7 to 16.9±2.9 % (p<0.01). Dabigatran also altered dynamics of TG with a prolongation of the lag-phase and a reductions in the maximal thrombin peak and potential of thrombin generation (p<0.01). In ROTEM, dabigatran significantly prolonged clotting time to 352±60 sec (p<0.01) and clot formation time to 312±76 sec (p<0.05). Idarucizumab completely reversed the alterations in all different biomarkers induced by dabigatran. Additionally, fibrin coverage and platelet deposition were restored to baseline values in flow studies. TG and ROTEM parameters also returned to normal values after idarucizumab. Reversal strategies with aPCC or PCC normalized and even over-compensated alterations in TG kinetics and partially improved alterations in ROTEM parameters caused by dabigatran. Interestingly, aPCC and PCC moderately improved the alteration in fibrin deposition caused by dabigatran in flow studies (15.7±8.2, 29.3±14.5, and 15.2±3.7 %, respectively for aPCCs 25, 75 or PCCs 70 IU/kg). However, levels of fibrin formation did not return to baseline values before dabigatran (67.2±32.5 %). rFVIIa showed only moderate effects on some of the biomarkers evaluated, though values were never restored to the baseline. CONCLUSIONS: Dabigatran (184 ng/mL) added to blood from healthy volunteers caused evident alterations in hemostasis parameters related to its recognized anticoagulant action. Procoagulant concentrates significantly compensated for the overall anti-hemostastic action of dabigatran. Overall, 75 U/kg aPCC seemed the more efficient nonspecific reversal therapy. In clear contrast with non specific procoagulant strategies, idarucizumab, the specific antidote to dabigatran completely reversed all alterations in coagulation parameters evaluated in circulating human blood and in assay systems. (Supported by SAF 2011-2814 and PI13/00517, Spanish Gov & FEDER) Disclosures van Ryn: Boehringer Ingelheim Pharma: Employment. Escolar:Boehringer Ingelheim Pharma: Investigator Sponsored Research Funding Other.

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The Resusciatative Fluid You Chose May Potentiate Bleeding.

Blood ◽

10.1182/blood.v104.11.1029.1029 ◽

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%) < HES (−8%)< 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 > 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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Fibrinogen: A Procoagulant and An Anticoagulant

Blood ◽

10.1182/blood.v118.21.384.384 ◽

2011 ◽

Vol 118 (21) ◽

pp. 384-384 ◽

Cited By ~ 1

Author(s):

Catherine J Rea ◽

Benny Sorensen ◽

Jørgen Ingerslev ◽

Peter Laursen

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Plasma Concentrations ◽

Lag Time ◽

Clot Formation ◽

Fibrinogen Concentration ◽

Clotting Time ◽

Dose Dependent ◽

Clot Stability

Abstract Abstract 384 Fibrinogen: A Procoagulant and an Anticoagulant Introduction: Bleeding occurs secondary to acquired fibrinogen deficiency but the effect of high fibrinogen is more controversial. Correlation between raised fibrinogen levels and venous or arterial thrombosis has been recorded. However, fibrinogen increases as an acute phase response and may be an innocent biomarker, detected at elevated levels in individuals with concomitant disease. Recent animal studies provide evidence that high fibrinogen does not trigger thrombosis per se, but enhances thrombotic occlusion of vessels following tissue injury. Aims: This study aims to investigate the effect of elevated levels of fibrinogen on thrombin generation and clot resistance to accelerated fibrinolysis. We hypothesised that fibrinogen promotes clot stability following a high tissue factor stimulus (TF), but will act as an anticoagulant following low TF stimulus. Method: Normal human plasma was spiked with fibrinogen to achieve final plasma concentrations of 2.7, 3.2, 3.7, 4.7, 5.7, 6.7, 8 and 11.7g/l. Coagulation was initiated with TF at variable dilutions (1:20000, 1: 500) plus calcium. To assess clot stability the same assay was performed with simultaneous addition of tissue plasminogen activator (t-Pa 0.75nmolar). Clot formation and lysis was recorded via light absorbance (FLUOstar Omega). Clot stability was also measured by whole blood thromboelastometry; citrate and CTI stabilized whole blood was drawn from a healthy individual and spiked with fibrinogen (calculated plasma concentrations: 3.2, 4, 3.9, 5.5, 9.9, 16 g/l). Coagulation was triggered with TF (1: 50000 or 1:500), calcium and of t-Pa (2nmolar). The area under elasticity curve (AUEC) at 90mins was the primary endpoint. Thrombin generation in plasma was performed in plasma following addition of fibrinogen using fluorogenic substrate and calcium (FluCä, Thrombinoscope BV, The Netherlands). Results:Plasma clot formation assay: Dose dependent shortening of clot time and time to peak turbidity were seen with increasing fibrinogen following a high TF stimulus (TF 1:500) (figure 1- panel A). Conversely, following a low TF stimulus increasing fibrinogen caused a lengthening of the clotting time. Plasma and whole blood lysis assays: With high TF stimulus, fibrinogen produced a dose-dependent increase in clot stability measures (AUC/AUEC) in both plasma and whole blood assays (figure 1-panel B). Following a low TF stimulus increases in the fibrinogen concentration resulted in suppressed clot stability. Thrombin generation: A decrease in total thrombin generation was seen with increasing fibrinogen (Figure 2) at both high and low TF levels. With high TF there was no alteration in lag-time, but with low TF stimulus the lag-time progressively lengthened as fibrinogen concentration increased. Discussion: Fibrinogen acts as a pro-coagulant by promoting clot formation and supports clot stability following a high TF stimulus. However, following a low TF stimulus elevated fibrinogen becomes an anticoagulant as demonstrated by prolonging clotting time and decreases clot stability in both plasma and whole blood. In conclusion, our data suggest that elevated fibrinogen per se is not thrombogenic. However, following a significant trauma resulting in a high tissue factor stimulus and high thrombin generation, fibrinogen acts predominantly as a pro-coagulant enhancing clot formation and supporting clot stability. This may protect against bleeding or contribute to pathological thrombotic events. In contrast, following a minor trauma prompting a minimal tissue factor stimulus, fibrinogen predominantly acts as an anticoagulant and may protect against thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Blood clotting in minimally altered whole blood

Blood ◽

10.1182/blood.v88.9.3432.bloodjournal8893432 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3432-3445 ◽

Cited By ~ 291

Author(s):

MD Rand ◽

JB Lock ◽

C van't Veer ◽

DP Gaffney ◽

KG Mann

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Antithrombin Iii ◽

Platelet Factor 4 ◽

Clot Formation ◽

Factor V ◽

Platelet Factor ◽

Typical Experiment ◽

Clot Time

The sequences of events regulating thrombin generation during tissue factor-initiated clotting in whole blood at 37 degrees C in which the contact pathway was suppressed with corn trypsin inhibitor are studied using quantitative Western blotting of factor V, prothrombin, platelet factor 4, antithrombin III, and fibrinogen. In addition, fibrinopeptide A (FPA), thrombin-antithrombin III (TAT) complex formation, and prothrombin fragment 1.2 (F1.2) were measured via commercially available enzyme-linked immunosorbent assays (ELISAs). In a typical experiment initiated with 40 pmol/L recombinant tissue factor, visual clot time (4.5 minutes), was preceded by significant cleavage of factor V resulting in 65% factor Va heavy-chain generation but only 10% light-chain formation. At this point, 50% of the platelet factor 4 is released, suggesting that half (approximately 700 pmol/L) of the platelet prothrombinase sites available have been generated. At clot time, approximately 15 nmol/L thrombin B-chain is present; however, analyses of FPA release demonstrate that only 15% of the thrombin is acting on fibrinogen. This thrombin is produced by the action of 7 pmol/L prothrombinase. The maximum rate of thrombin production is reached well after clot time and is consistent with the presence of approximately 150 pmol/L prothrombinase (at about 7 minutes). These results suggest that factor Xa is the limiting factor for thrombin generation. After 60 minutes, 75% of the initial prothrombin (1.24 mumol/L) is consumed yielding 400 nmol/ L prethrombin 2 and 360 nmol/l thrombin (B-chain) products. The sum of these values (800 nmol/L) is similar to the (corrected) F1.2 concentration determined by ELISA. The incomplete cleavage of prothrombin indicates both the prothrombinase complex and the formation of prothrombinase are inhibited in the reaction. TAT complex measured by ELISA is almost equivalent to B-chain concentration, but sodium dodecyl sulfate stable thrombin-antithrombin III complexes are not observed until well after clot formation and are never equivalent to ELISA-TAT values. At the point of clot formation, 80% of the fibrinogen is depleted from the fluid phase, whereas only 35% to 45% of the FPA is released, suggesting a significant incorporation of uncleaved fibrinogen into the initial clot formed.

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