Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3008-3013 ◽  
Author(s):  
Alisa S. Wolberg ◽  
Dougald M. Monroe ◽  
Harold R. Roberts ◽  
Maureane Hoffman
Keyword(s):  
Plasma Levels ◽  
Thrombin Generation ◽  
Fibrin Clot ◽  
Length Ratio ◽  
Dependent Manner ◽  
Thrombotic Risk ◽  
Increased Risk ◽  
Clot Structure ◽  
Dose Dependent ◽  
Fibrin Clots

AbstractIndividuals with elevated prothrombin levels are at increased risk of venous thrombosis. To understand the mechanism behind this observation, we studied the effect of prothrombin concentration on thrombin generation and fibrin clot structure. The pattern of thrombin generation was directly related to the prothrombin level at all concentrations tested. From 0% to 300% of normal plasma levels of prothrombin, increasing the prothrombin concentration increased the initial rate, peak, and total amount of thrombin generated. Importantly, fibrin clot structure was also affected by the prothrombin concentration. Fibrin clots made from prothrombin concentrations less than 10% of plasma levels were weak and poorly formed. Fibrin clots made at 10% to 100% of plasma levels of prothrombin had similar fiber structures (mass-to-length ratio; μ). However, the fiber mass-to-length ratio decreased with increasing prothrombin levels more than 100% of plasma levels, in a dose-dependent manner. These results suggest that increased levels of prothrombin alter thrombin generation and clot structure. Specifically, elevated prothrombin levels produce clots with reduced fibrin mass-to-length ratios compared with normal clots. We hypothesize that this alteration in fibrin clot structure is an important determinant of the risk of thrombosis.

How can We Predict Efficacy of rFVIIa in Hemophilia Patients with Inhibitors?

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3487-3487
Author(s):  
Yesim Dargaud ◽  
Jean C Bordet ◽  
Chantal Huchon ◽  
Claude Negrier
Keyword(s):  
Thrombin Generation ◽  
Fibrin Clot ◽  
Bypassing Agents ◽  
The Stability ◽  
Clot Structure ◽  
Dose Dependent ◽  
Clot Stability ◽  
Scanning Electron ◽  
Fibrin Clots

Abstract Abstract 3487 Poster Board III-424 Hemophilia patients with inhibitors are treated with bypassing agents for which hemostatic efficacy is unpredictable. While both activated prothrombin complex concentrate and recombinant activated factor VII (rFVIIa) have demonstrated excellent safety profiles, neither product is a universal hemostatic agent and the variability of response to bypassing agents complicates the treatment in these patients. Moreover, the lack of a validated laboratory assay to measure the effectiveness of bypassing agents dramatically limits the optimisation of treatment strategies. As the final enzyme generated by bypassing agents is thrombin, thrombin generation assay (TGA) could theoretically be used for monitoring rFVIIa. However, TGA does not reflect the stability of the fibrin clot and its resistance to fibrinolysis which are essential parameters of hemostasis. We have therefore evaluated the use of an additional method that might provide complementary information on fibrin clot structure and stability, and would allow a better prediction of the biological efficacy of rFVIIa. In the absence of FVIII/FIX, fibrin fibres are abnormally thick and clots are overly susceptible to fibrinolysis. After treatment with rFVIIa, clots are less porous and fibrin fibres are thinner as assessed using scanning electron microscopy. Using whole blood thromboelastography (TEG) measuring viscoelastic changes of fibrin throughout clot initiation, formation and fibrinolysis, we developed an in vitro model to assess fibrin clot stability and resistance to fibrinolysis. The aim of the present study was to evaluate the correlation between the modifications of the fibrin clot structure and the stability of the fibrin clots obtained in the presence of rFVIIa. METHODS The in vitro effect of rFVIIa was tested in 6 severe hemophilia A patients at doses of 90 – 180 – 270 μg/kg. Thrombin generation (TG) was measured in platelet rich plasma using the CAT method in the presence of TF 1pM. After addition of rFVIIa, the improvement in TG capacity was compared to normal values obtained from 80 control males. Fibrin clots obtained from the TG measurements were studied by scanning electron microscopy (SEM) and fibrin diametres were measured (700 measurements on each sample). In the same samples, the stability of fibrin clots obtained before and after addition of rFVIIa was assessed using TEG-5000™. Clot resistance to fibrinolysis was recorded in the presence of TF 0.5pM and tPA 0.125μM. TEG-tPA and SEM results were compared to those obtained in 30 healthy control males. RESULTS A dose dependent increase of TG was observed in the presence of increasing doses of rFVIIa (p<0.0001; ANOVA). In the presence of rFVIIa 90μg/kg, TG capacity was significantly improved in all patients (p=0.0023; Mann Whitney), and was completely normalized in 4 patients while 2 others needed higher doses of rFVIIa to normalize their TG. The fibrin fibre diameters were thicker (217±16 nm; p<0.0001) in all hemophilia patients in comparison with controls (170±24 nm). After addition of rFVIIa 90μg/kg, the fibrin clot structure was modified and the diameter of fibrin fibres was dramatically decreased in all patients (184±11 nm; p=0.006). A further improvement of fibrin clot structure was observed with rFVIIa 180μg/kg in only one patient. TEG-tPA showed a dose-dependent improvement of fibrin clot stability in the presence of rFVIIa (p<0.0001; ANOVA). A reverse correlation was observed between fibrin fibre diametres and resistance of fibrin clots to fibrinolysis (r=-0.68, p=0.001; Spearman test). CONCLUSION This data demonstrates a statistically significant correlation between clot structure and its stability. The combined use of TGA with TEG-tPA may allow physicians to better evaluate the individual response of patients to bypassing agents. The clinical validity of the minimal individual dose of rFVIIa normalizing both TGA and TEG-tPA needs to be verified in clinical studies. Disclosures: No relevant conflicts of interest to declare.

Fibrin clot properties and haemostatic function in men and women with type 1 diabetes

2015 ◽  
Vol 113 (02) ◽  
pp. 312-318 ◽  
Author(s):  
Gun Jörneskog ◽  
Anna Ågren ◽  
Per-Eric Lins ◽  
Håkan Wallén ◽  
Aleksandra Antovic ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Type 1 Diabetes ◽  
Thrombin Generation ◽  
Microvascular Complications ◽  
Fibrin Clot ◽  
Men And Women ◽  
Increased Risk ◽  
Fibrin Clots

SummaryThe increased risk of vascular complications in type 1 diabetes may in part be explained by changes in haemostatic function. In the present study, we investigated the fibrin clot properties in patients with type 1 diabetes in relation to sex and microvascular complications. The study included 236 patients (107 women) aged between 20–70 years and without any history of cardiovascular disease. Fibrin clot properties, assessed by determination of the permeability coefficient (Ks) and turbidimetric clotting and lysis assays, did not differ between men and women. Compared with men, women had worse glycaemic control as well as higher levels of prothrombin fragment 1+2 and peak thrombin generation in vitro, indicating increased thrombin generation both in vivo and in vitro. Subgroup analyses of patients younger than 30 years revealed less permeable fibrin clots and prolonged lysis time in females compared with age-matched men. Patients with microvascular complications had higher fibrinogen concentrations and denser and less permeable fibrin clots. Thus, we conclude that in vitro fibrin clot properties in patients with type 1 diabetes without cardiovascular disease are not different between the sexes, but associate with prevalence of microvascular complications. Tighter fibrin clot formation in younger women, as suggested by our results, may affect their future cardiovascular risk and should be investigated in a larger population.

Improvement of fibrin clot structure after factor VIII injection in haemophilia A patients treated on demand

2014 ◽  
Vol 111 (04) ◽  
pp. 656-661 ◽  
Author(s):  
Danijela Mikovic ◽  
Ivo Elezovic ◽  
Michael Zabczyk ◽  
Kjell Hutenby ◽  
Jovan Antovic ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Fibrin Clot ◽  
Haemophilia A ◽  
Sem Images ◽  
On Demand ◽  
Before And After ◽  
Fibrinolysis Inhibitor ◽  
Clot Structure ◽  
Fibrin Structure ◽  
Fibrin Clots

SummaryPatients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.

scholarly journals Polyphosphate enhances fibrin clot structure

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2810-2816 ◽  
Author(s):  
Stephanie A. Smith ◽  
James H. Morrissey
Keyword(s):  
Fibrin Clot ◽  
Factor Xiiia ◽  
Clot Lysis ◽  
Factor V ◽  
Scanning Electron Micrographs ◽  
Dependent Manner ◽  
Dense Granules ◽  
Contact Pathway ◽  
Clot Structure ◽  
Fibrin Clots

Abstract Polyphosphate, a linear polymer of inorganic phosphate, is present in platelet dense granules and is secreted on platelet activation. We recently reported that polyphosphate is a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation. Because polyphosphate did not alter thrombin clotting times, it appeared to exert all its procoagulant actions upstream of thrombin. We now report that polyphosphate enhances fibrin clot structure in a calcium-dependent manner. Fibrin clots formed in the presence of polyphosphate had up to 3-fold higher turbidity, had higher mass-length ratios, and exhibited thicker fibers in scanning electron micrographs. The ability of polyphosphate to enhance fibrin clot turbidity was independent of factor XIIIa activity. When plasmin or a combination of plasminogen and tissue plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of polyphosphate exhibited prolonged clot lysis times. Release of polyphosphate from activated platelets or infectious microorganisms may play an important role in modulating fibrin clot structure and increasing its resistance to fibrinolysis. Polyphosphate may also be useful in enhancing the structure of surgical fibrin sealants.

scholarly journals Paternal smoking is associated with an increased risk of pregnancy loss in a dose-dependent manner: a systematic review and meta-analysis

F&S Reviews ◽  
2021 ◽  
Author(s):  
Nadia A. du Fossé ◽  
Marie-Louise P. van der Hoorn ◽  
Nina H. Buisman ◽  
Jan M.M. van Lith ◽  
S askia le Cessie ◽  
...  
Keyword(s):  
Systematic Review ◽  
Pregnancy Loss ◽  
Meta Analysis ◽  
Dependent Manner ◽  
Increased Risk ◽  
Dose Dependent ◽  
Paternal Smoking

scholarly journals Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions

2013 ◽  
Vol 109 (02) ◽  
pp. 221-228 ◽  
Author(s):  
Keisuke Soya ◽  
Fumiko Terasawa ◽  
Nobuo Okumura
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Fibrin Clot ◽  
Fibrinopeptide A ◽  
Thrombin Cleavage ◽  
No Release ◽  
Per Se ◽  
Clot Structure ◽  
Dose Dependent ◽  
Scanning Electron

SummaryFibrin polymerisation is mediated by interactions between knobs ‘A’ and ‘B’ exposed by thrombin cleavage, and holes ‘a’ and ‘b’. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H-fibrinogen with impaired hole ‘a’. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H-fibrinogen, we examined two variant fibrinogens with substitutions altering knob ‘A’, Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γD364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H-fibrinogen. No variants polymerised with batroxobin, which exposed only knob ‘A’. The inhibition of variant fibrinogens’ polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes ‘b’. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C-fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.

Inflammation and thrombosis in diabetes

2011 ◽  
Vol 105 (S 06) ◽  
pp. S43-S54 ◽  
Author(s):  
Katharina Hess ◽  
Peter Grant
Keyword(s):  
Plaque Rupture ◽  
Thrombus Formation ◽  
Atherosclerotic Lesion ◽  
Coagulation Factors ◽  
Fibrin Clot ◽  
Thrombotic Risk ◽  
Cardiovascular Morbidity And Mortality ◽  
Increased Risk ◽  
Patients With Diabetes ◽  
Inflammatory State

SummaryPatients with diabetes mellitus are at increased risk of cardiovascular morbidity and mortality. Atherothrombosis, defined as atherosclerotic lesion disruption with superimposed thrombus formation, is the most common cause of death among these patients. Following plaque rupture, adherence of platelets is followed by local activation of coagulation, the formation of a cross-linked fibrin clot and the development of an occlusive platelet rich fibrin mesh. Patients with diabetes exhibit a thrombotic risk clustering which is composed of hyper-reactive platelets, up regulation of pro-thrombotic markers and suppression of fibrinolysis. These changes are mainly mediated by the presence of insulin resistance and dysglycaemia and an increased inflammatory state which directly affects platelet function, coagulation factors and clot structure. This prothrombotic state is related to increased cardiovascular risk and may account for the reduced response to antithrombotic therapeutic approaches, underpinning the need for adequate antithrombotic therapy in patients with diabetes to reduce their cardiovascular mortality.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

scholarly journals Synergistic Effect of Bypassing Agents and Sequence Identical Analogue of Emicizumab and Fibrin Clot Structure in the In Vitro Model of Hemophilia A

TH Open ◽  
2020 ◽  
Vol 04 (02) ◽  
pp. e94-e103
Author(s):  
Yanan Zong ◽  
Aleksandra Antovic ◽  
Nida Mahmoud Hourani Soutari ◽  
Jovan Antovic ◽  
Iva Pruner
Keyword(s):  
In Vitro Model ◽  
Hemophilia A ◽  
Fibrin Clot ◽  
Substantial Improvement ◽  
Activated Prothrombin Complex Concentrate ◽  
Bypassing Agents ◽  
Vitro Model ◽  
Clot Structure ◽  
Fibrin Clots

AbstractDevelopment of inhibitors to factor VIII (FVIII) occurs in approximately 30% of severe hemophilia A (HA) patients. These patients are treated with bypassing agents (activated prothrombin complex concentrate [aPCC] and recombinant activated FVII-rFVIIa). Recently, a bispecific FIX/FIXa- and FX/FXa-directed antibody (emicizumab) has been approved for the treatment of HA patients with inhibitors. However, the data from clinical studies imply that coadministration of emicizumab and bypassing agents, especially aPCC, could have a thrombotic effect.This study was aimed to address the question of potential hypercoagulability of emicizumab and bypassing agents' coadministration, we have investigated fibrin clot formation and structure in the in vitro model of severe HA after adding sequence-identical analogue (SIA) of emicizumab and bypassing agents.Combined overall hemostasis potential (OHP) and fibrin clot turbidity assay was performed in FVIII-deficient plasma after addition of different concentrations of SIA, rFVIIa, and aPCC. Pooled normal plasma was used as control. The fibrin clots were analyzed by scanning electron microscopy (SEM).OHP and turbidity parameters improved with the addition of aPCC, while therapeutic concentrations of rFVIIa did not show substantial improvement. SIA alone and in combination with rFVIIa or low aPCC concentration improved OHP and turbidity parameters and stabilized fibrin network, while in combination with higher concentrations of aPCC expressed hypercoagulable pattern and generated denser clots.Our in vitro model suggests that combination of SIA and aPCC could potentially be prothrombotic, due to hypercoagulable changes in fibrin clot turbidity and morphology. Additionally, combination of SIA and rFVIIa leads to the formation of stable clots similar to normal fibrin clots.

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Blood ◽  
2011 ◽  
Vol 118 (14) ◽  
pp. 3942-3951 ◽  
Author(s):  
Joke Konings ◽  
José W. P. Govers-Riemslag ◽  
Helen Philippou ◽  
Nicola J. Mutch ◽  
Julian I. Borissoff ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Direct Interaction ◽  
Fibrin Clot ◽  
Fiber Density ◽  
Contact Pathway ◽  
Clot Structure ◽  
Fibrin Structure

Abstract Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.

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