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

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.

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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 ◽

10.1055/s-0040-1710032 ◽

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.

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

Blood ◽

10.1182/blood-2002-08-2527 ◽

2003 ◽

Vol 101 (8) ◽

pp. 3008-3013 ◽

Cited By ~ 113

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.

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

Thrombosis and Haemostasis ◽

10.1160/th12-09-0684 ◽

2013 ◽

Vol 109 (02) ◽

pp. 221-228 ◽

Cited By ~ 8

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.

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

Thrombosis and Haemostasis ◽

10.1160/th14-05-0404 ◽

2015 ◽

Vol 113 (02) ◽

pp. 312-318 ◽

Cited By ~ 5

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.

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

Thrombosis and Haemostasis ◽

10.1160/th13-06-0479 ◽

2014 ◽

Vol 111 (04) ◽

pp. 656-661 ◽

Cited By ~ 7

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.

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Difference in Fibrin Clot Structure in Haemophilia A and Haemophilia B

Blood ◽

10.1182/blood.v128.22.563.563 ◽

2016 ◽

Vol 128 (22) ◽

pp. 563-563

Author(s):

Vincent Luong ◽

Nida Soutari ◽

Maria Berndtsson ◽

Margareta Holmström ◽

Jovan P. Antovic ◽

...

Keyword(s):

Research Funding ◽

Hemophilia A ◽

Coagulation Factor ◽

Fibrin Clot ◽

Hemophilia B ◽

Severe Bleeding ◽

Factor Level ◽

Clot Structure ◽

Fibrin Clots

Abstract Introduction: Hemophilia A and Hemophilia B are X-linked bleeding disorders characterized by deficiency of coagulation factor VIII and IX respectively. The disorders have traditionally been considered to display identical symptoms. However, recent clinical data suggest that hemophilia B has a less severe phenotype than hemophilia A in patients with similar levels of their deficient factor. There is a distinct lack of laboratory studies comparing the hemostatic potential in patients with the same severity grade of Hemophilia A and Hemophilia B. We assessed fibrin clot quality in Hemophilia A and Hemophilia B patient plasma as a possible explanation to the milder bleeding phenotype in patients with Hemophilia B. To achieve this a liquid permeation technique was used, which has been used to investigate fibrin networks in previous in vitro studies (He et. al. Blood Coagul Fibrinolysis 2005). Methods: 20 patients with hemophilia A and 28 with hemophilia B were considered for inclusion in the study. Patients were recruited from centers in Stockholm and Belgrade and were thereafter matched according to their deficient factor level. Hemophilia diagnosis and informed consent formed the basis for study inclusion and exclusion criteria were lack of consent and inability to provide a matched patient from the other group. 17 hemophilia A and 17 hemophilia B patients were included in the study. The patient matching according to factor level was tested with Spearman's rank-order correlation, yielding a correlation coefficient of 0.999 (p<0.001). In vitro fibrin clots were created from centrifugated patient plasma, CaCl2 and bovine thrombin. They were then permeated with a percolating buffer with a pre-determined viscosity (Tris-hydroxymethyl aminomethane, imidazole, NaCl and trasylol) under three different pre-determined hydrostatic pressures. Fibrin clot permeability was calculated in Darcy constants (Ks) using the equation: Ks = (Q x L x h) / (t x A x ΔP). where Q is the elution volume (cm3), t is the buffer percolation time (seconds), h is the viscosity (poise, dyne x s cm-2), L is the fibrin clot length (cm), A is the area of the fibrin clot (cm2) and ΔP is the difference of pressure (dyne cm-2). A high Ks represents high permeability, which is interpreted as porous fibrin clots. In contrast low Ks values represent low permeability, which is interpreted as low porosity. Results: Ks was higher in hemophilia A than in hemophilia B. Mean Ks for hemophilia A was 12.28 ± 5.92 and for hemophilia B 6.16 ± 3.63 (p<0.0005). Subanalysis of moderate and severe hemophilia patients (8 matched pairs, 16 patients) showed higher mean Ks in hemophilia A. Mean Ks of the hemophilia A group was 14.90 ± 6.88 and for the hemophilia B group 5.78 ± 3.86, p=0.039. Conclusion: Fibrin clot structure exhibits less permeability in hemophilia B than hemophilia A, which may be one of the explanations to the milder bleeding symptoms observed in hemophilia B at the same level of deficient factor. This finding is in accordance with clinical studies showing that HB has a less severe bleeding phenotype than HA. Disclosures Antovic: Baxter Healthcare Corporation: Honoraria, Research Funding; Siemens: Honoraria; Stago: Honoraria; Sysmex: Honoraria; Novo Nordisk: Honoraria; Roche: Honoraria. Chaireti:Baxalta: Research Funding.

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Incorporation of α2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients

Biomolecules ◽

10.3390/biom11030347 ◽

2021 ◽

Vol 11 (3) ◽

pp. 347

Author(s):

Zsuzsa Bagoly ◽

Barbara Baráth ◽

Rita Orbán-Kálmándi ◽

István Szegedi ◽

Réka Bogáti ◽

...

Keyword(s):

Ischemic Stroke ◽

Acute Ischemic Stroke ◽

Intracranial Hemorrhage ◽

Intravenous Thrombolysis ◽

Fibrin Clot ◽

Clot Lysis ◽

Stroke Patients ◽

Plasmin Inhibitor ◽

Fibrin Clots

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

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Effect of Photosensitization Mediated by Curcumin on Carotenoid and Aflatoxin Content in Different Maize Varieties

Applied Sciences ◽

10.3390/app11135902 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5902

Author(s):

Rafael Nguenha ◽

Maral Seidi Damyeh ◽

Anh D. T. Phan ◽

Hung T. Hong ◽

Mridusmita Chaliha ◽

...

Keyword(s):

Health Risks ◽

Carotenoid Content ◽

Naturally Occurring ◽

Maize Varieties ◽

Food And Feed ◽

Maize Kernels ◽

Dose Dependent ◽

Preservation Technique ◽

Scanning Electron

Mycotoxins are naturally occurring toxins produced by certain types of fungi that contaminate food and feed, posing serious health risks to human and livestock. This study evaluated the combination of blue light with curcumin to inactivate Aspergillus flavus spores, its effect on aflatoxin B1 (AFB1) production and maintaining carotenoid content in three maize varieties. The study was first conducted in vitro, and the spore suspensions (104 CFU·mL−1) were treated with four curcumin concentrations (25 and 50 µM in ethanol, 1000 and 1250 µM in propylene glycol) and illuminated at different light doses from 0 to 130.3 J·cm−2. The photoinactivation efficiency was light-dose dependent with the highest photoinactivation of 2.3 log CFU·mL−1 achieved using 1000 µM curcumin at 104.2 J·cm−2. Scanning electron microscopy revealed cell wall deformations as well as less density in photosensitized cells. Photosensitization of maize kernels gave rise to a complete reduction in the viability of A. flavus and therefore inhibition of AFB1 production, while no significant (p > 0.05) effect was observed using either light or curcumin. Moreover, photosensitization did not affect the carotenoids in all the studied maize varieties. The results suggest that photosensitization is a green alternative preservation technique to decontaminate maize kernels and reduce consumer exposure to AFB1 without any effect on carotenoid content.

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

Blood ◽

10.1182/blood-2011-03-339572 ◽

2011 ◽

Vol 118 (14) ◽

pp. 3942-3951 ◽

Cited By ~ 84

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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