Difference in Fibrin Clot Structure in Haemophilia A and Haemophilia B

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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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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FranceCoag Network: A National Multicenter Prospective Cohort for Congenital Bleeding Disorders.

Blood ◽

10.1182/blood.v106.11.4094.4094 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4094-4094

Author(s):

Edith Fressinaud ◽

Jocelyne Dieval ◽

Valerie Gay ◽

Viviane Guerin ◽

Thierry Lambert ◽

...

Keyword(s):

Public Health ◽

Hemophilia A ◽

Coagulation Factor ◽

Steering Committee ◽

Hemophilia B ◽

Cumulative Exposure ◽

Severe Bleeding ◽

Public Health Agencies ◽

National Cohort

Abstract The FranceCoag Network is a prospective national cohort of patients affected with hemophilia and severe hereditary hemorrhagic diseases except platelet disorders. It has been set up in France since January 2003 as replacement of a previous project only dedicated to hemophilia. The objectives are to know the exhaustive number, type and characteristics of these patients registered in hemophilia centers. Inclusion criteria are: FVIII or IX defect (<30%), severe defect (<10%) in FII, V, VII, X, XI, XIII or FI (< 0.1 g/l) or severe VWD. Once per year centers send datas on severe bleeding episodes, surgery, replacement therapy i.e. type of concentrate, units, place of treatment and main complications (including new inhibitors). These data are collected and monitored by a coordinating center attached to a public health institution (InVS). Direct transmission of datas via a highly protected web site is foreseen for the end of 2005. The follow-up of the study is ensured by a steering committee (regional coordinators, clinicians, committed health institutions, representatives of patients association) periodically extended to scientific experts and other public health agencies. Beside its epidemiologic interest, this cohort will also promote research projects on this population such as Pups cohort (defined as severe hemophilia A or B patients with none or few cumulative exposure days at the enrolment). On July 2005, 3469 patients were included by 37 centers. The diagnosis are hemophilia A n=2594 (75%); hemophilia B n=539 (15%), VWD n=239 (7%), severe defect in other coagulation factor: n= 97 (3%). At their inclusion, 373 patients (11%) were infected by HIV1, 1383 (40%) had a positive HCV serology, while 365 patients (10%) were co infected by HIV1 and HCV. Among this cohort, 45 deaths were observed since 1994, which represent 4.5 deaths per 1 000 years-patients (95% CI: [3.3–6.0]). Deaths were related to AIDS or HCV in 19 cases. Inhibitors was observed in 388 patients (11%) (hemophilia A: n=369, hemophilia B n=15, VWD n=2, FXIII defect n= 1, FV defect n=1). The incidence of inhibitor during the follow-up was 9.4 cases per 1000 person-years.

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

Blood ◽

10.1182/blood.v114.22.3487.3487 ◽

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.

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Thrombin Generation As a Promising Biomarker for the Prediction of Bleeding Phenotype in Patients with Non-Severe Hemophilia

Blood ◽

10.1182/blood-2018-99-119198 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3788-3788

Author(s):

Fadi Nossair ◽

Nina Hwang ◽

Vanessa Salinas ◽

Nicole Crook ◽

Jacqueline Limjoco ◽

...

Keyword(s):

Research Funding ◽

Hemophilia A ◽

Hemophilia B ◽

P Value ◽

Severe Hemophilia ◽

Factor Activity ◽

Factor Level ◽

Bleeding Score ◽

Baseline Factor

Abstract Background: Non-severe hemophilia A and B account for 50% of patients with hemophilia, in which factor level does not consistently correlate with bleeding phenotype. Clinical decision-making in regards to timely prophylaxis initiation and tailored surgical management could be informed by a biomarker that is more predictive of bleeding phenotype. We hypothesized that a global method to assess clotting potential, such as thrombin generation (TG), is more predictive of bleeding phenotype than factor level. Objectives: Determine the ability of TG, as compared to standard baseline factor activity, to differentiate bleeding phenotype severity in patients with non-severe hemophilia. Methods: Subjects were recruited from two hemophilia treatment centers (HTCs): Rady Children's Hospital San Diego (RCHSD) and Center for Inherited Blood Disorders (CIBD). Subjects were eligible for enrollment if they were at least one year of age and had a diagnosis of non-severe congenital hemophilia A or B, or were genetically confirmed or suspected female carriers. All enrolled patients or their parents completed the standardized, self-administered pediatric bleeding questionnaire (Self-PBQ) or bleeding assessment tool (Self-BAT). Clinical and laboratory information were extracted from the medical chart, including age at diagnosis, bleeding event history, past surgical history, treatment history and factor VIII or IX gene analysis. Validation of self-reported bleeding symptoms was performed using chart-derived data. Bleeding phenotype was assessed using standard calculation of the bleeding score, as defined by the respective validated self-reported tools. For the purpose of this analysis, we defined a high bleeding score as 13 or more, while a low bleeding score was defined as 12 or less. After a washout period of 5x the standard half-life of the administered factor product, blood samples were collected at time of enrollment. Platelet poor plasma was obtained according to a strict protocol to minimize pre-analytical variables. TG was measured by means of the calibrated automated thrombogram, with three different reagents: low (1 pM of tissue factor [TF]), regular (5 pM of TF) and High (20 pM of TF). The following TG parameters were evaluated: Peak TG (Peak), estimated thrombin potential (ETP) and velocity index (VI). Results: Eighty-one subjects were enrolled. The median age of our cohort was 15.6 years (IQR 21.2, range 4.9 - 59.8), with a slight female predominance (51%) due to inclusion of female carriers. The median follow-up period at the HTC was 5.3 years (IQR 7.5 years), with the majority having follow-up for at least three years. Enrolled patients had the following diagnoses: mild hemophilia A (70%), mild hemophilia B (3%), moderate hemophilia A (13%), moderate hemophilia B (3%), hemophilia A carriers with normal factor VIII levels (10%) and hemophilia B carriers with normal factor IX levels (1%). Median age at diagnosis was 8 years (IQR 26.3, range 0.1 - 56.1), which strongly correlated lower baseline factor activity (r=0.573, p-value < 0.0001). The median baseline factor activity was 21% (IQR 26, range 2 - 249). Factor exposure occurred in 46% of patients (n=37), of which 5 patients were on prophylaxis at time of enrollment. Chronic arthropathy was present in 2 patients (one with mild hemophilia A and one with moderate hemophilia A) and none of the patients had a history of an inhibitor. The majority of patients had a low bleeding score (74% vs. 26%). Baseline factor level and TG values obtained with regular reagent (5 pM of TF) showed significant correlation with bleeding score (r = -0.229 to -0.237, p-value < 0.05), while values obtained with other reagents did not show a significant correlation. Sensitivity/specificity analysis revealed the following optimal cutoff values for differentiating between bleeding severities, as obtained from TG with regular reagent: ETP (<1240 nM min), Peak (<130 nM) and VI (<23 nM/min), with analysis results as shown in Table 1. Conclusion: Even though both TG and baseline factor level had comparable correlation with bleeding severity, all TG values with 5 pM TF showed a much higher sensitivity outcome and greater ability to differentiate between bleeding severities in this population. This approach shows potential for predicting bleeding severity in patients with non-severe hemophilia and should be validated in long-term prospective studies. Disclosures Nossair: Novo Nordisk: Other: Conference - Haemophilia Acadamy; Novo Nordisk: Research Funding. Hwang:BPI: Consultancy; Bayer: Consultancy; Hema Biologics: Consultancy; Shire: Consultancy; Bioverativ: Other: PI in clinical research study. Thornburg:ATHN: Research Funding; Bayer Pharmaceuticals: Research Funding; Biomarin: Consultancy; CSL Behring: Research Funding; Bioverativ: Consultancy; Genentech: Speakers Bureau; Octapharma: Research Funding; NovoNordisk: Research Funding; Shire: Research Funding; Johns Hopkins All Children's Hospital: Research Funding; Bluebird Bio: Consultancy.

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Recombinant Porcine Factor VIII Corrects Thrombin Generation and Improves Clot Structure In Vitro in Plasma Containing Anti-Factor VIII Inhibitory Antibodies

Blood ◽

10.1182/blood.v118.21.2282.2282 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2282-2282 ◽

Cited By ~ 1

Author(s):

Claude Negrier ◽

Shannon L. Meeks ◽

Johannes Oldenburg ◽

Uri Martinowitz ◽

Jean-Claude Bordet ◽

...

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Inhibitory Activity ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Cross Reactivity ◽

Advisory Committees ◽

Clot Structure

Abstract Abstract 2282 Introduction: Treatment of bleeding episodes in patients with hemophilia A who have developed inhibitory antibodies can be challenging. Using human factor VIII (FVIII) and, historically, porcine FVIII in patients with a low inhibitor titer are therapeutic options, and provide ease of monitoring. A B-domain deleted recombinant porcine FVIII (rpFVIII; OBI-1), which may possess low cross-reactivity to anti-human FVIII antibodies, is being investigated for the treatment of bleeding episodes in individuals with congenital hemophilia A and inhibitors, and in those with acquired hemophilia. The in vitro capacity of this molecule to correct hemostasis has been further characterized. Methods: This is an international, multicenter in vitro study. Individuals with hemophilia A and inhibitor antibodies were recruited during routine out-patient visits between January 2011 and March 2011. Written and signed informed consent was obtained prior to venepuncture. Blood was obtained from volunteers with congenital hemophilia A and inhibitors attending routine visits at participating hemophilia treatment centers. A single blood sample was obtained from consenting individuals under protocols approved by Institutional Review Boards/Ethical Committees. In vitro spiking experiments with OBI-1 were conducted using FVIII-deficient plasma with and without anti-FVIII inhibitory activity. Three control inhibitor plasmas were provided, composed of FVIII deficient plasma to which the anti-C1 monoclonal antibody (MAb) to human FVIII (Sanquin, Amsterdam, the Netherlands) was added at two concentrations to reach anti-human FVIII inhibitory activity of 4.9 Bethesda Units (BU)/mL and 32.8 BU/mL with anti-porcine anti-FVIII inhibitory activity of 2.7 BU/mL and 19.1 BU/mL, respectively; and FVIII deficient plasma to which “polyclonal” mixture of the anti-C1 MAb, along with an anti-A2 and 2 anti-C2 MAbs was added. Plasma from eight patients with hemophilia A and inhibitors was tested. Hemostatic correction by OBI-1 was assessed by thrombin generation measurement (Calibrated Analytical Thrombography assay, Synapse BV, Maastricht, The Netherlands) and clot structure using electron microscopy. Epitope mapping of the inhibitor patient plasma was undertaken at a central laboratory (Atlanta, Georgia, USA) using an Enzyme-Linked Immunosorbent Assay (ELISA) with human/porcine FVIII hybrids as the antigen. Results: The results showed a dose-dependent and anti-porcine titer dependent correction of thrombin generation parameters (peak and ETP) with OBI-1 at concentrations equivalent to 100 IU/dL, 200 IU/dL, and 400 IU/dL, which paralleled a correction of the clot structure (number and diameter of fibrin fibres). These results were only dependent on the anti-porcine titer. In samples with high titers of anti-porcine inhibitor (>10 BU), little or no restoration of the diminished thrombin generation was observed when various OBI-1 concentrations were added to the plasma. In the plasmas with high anti-human titers (≥10 BU/mL) the dominant epitope was C2 in 3 plasmas, A2 in 1 plasma, and indeterminate in 3 plasmas. The plasmas with no restoration of the thrombin generation with even the highest dose of OBI-1 all had antibody detected to more than one domain of FVIII or were not able to be mapped due to high porcine cross-reactivity. Conclusion: In vitro data obtained with spiking experiments using OBI-1 indicate that it has the potential to correct surrogate markers of hemostasis depending on the anti-porcine FVIII titer which may translate into in vivo effectiveness. Further investigation into the epitope specificity of responsive and non-responsive inhibitor plasmas correlation with effectiveness is warranted. Disclosures: Negrier: Inspiration Biopharmaceuticals: Honoraria, Research Funding. Meeks:Inspiration Biopharmaceuticals: Research Funding. Oldenburg:SOBI: Membership on an entity's Board of Directors or advisory committees; Catalyst: Membership on an entity's Board of Directors or advisory committees; Inspiration: Consultancy, Honoraria, Research Funding; LFB: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Honoraria; Octapharma: Consultancy, Honoraria, Research Funding. Bordet:Inspiration Biopharmaceuticals: Research Funding. Poetzsch:Inspiration Biopharmaceuticals: Research Funding. Al Dieri:Synapse BV: Employment. Dargaud:Inspiration Biopharmaceuticals: Research Funding. Hemker:Synapse BV: Employment. Eckmann:Sanquin Diagnostic Services: Employment. Gomperts:Inspiration Biopharmaceuticals: Employment. Lee:Inspiration Biopharmaceuticals: Employment.

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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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Impact of Systematic Pharmacokinetic (PK) Profiles in a Cohort of 29 Patients Treated with Conventional and Extended-Half Life (EHL) Products

Blood ◽

10.1182/blood-2019-132199 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1123-1123

Author(s):

Teresa Ceglie ◽

Berardino Pollio ◽

Irene Ricca ◽

Maria Messina ◽

Claudia Linari ◽

...

Keyword(s):

Hemophilia A ◽

Factor Ix ◽

Hemophilia B ◽

Null Mutation ◽

Severe Hemophilia ◽

Factor Level ◽

On Demand ◽

Active Life ◽

Genetic Assessment

Introduction. Prophylaxis with factor concentrates reduces bleeding events and improves quality of life for adults and children with severe hemophilia. However, the optimal dosing and infusion frequency is not yet established. Integration of PK data into decision making is gaining support, in particular at the transition between conventional and EHL products. Here we report about 29 PK data of patients affected by hemophilia treated at our centre since childhood. Improved quality of life was our first aim, supposed that decreasing frequency of infusions or increasing the target through factor level allows a more active life without increased risk of bleeding. Patients' characteristics and methods. 18 patients (62%) were ≤ 18 years of age at PK time. 16 were affected by severe hemophilia A, 5 by moderate hemophilia A, 6 by severe hemophilia B and 2 by moderate hemophilia B. At PK time, 28 patients were on prophylaxis and 1 was on demand with recombinant factor IX. Median age at onset of prophylaxis was 9 years (range 3 months-38 years). Genetic assessment was available in 24 patients. Of these, 37.5% and 62.5% were carriers of null and not null mutations respectively. 4 patients were undergone to PK with standard products (1 Octocog alfa, 1 Simoctocog alfa, 1 Octocog alfa-Kovaltry®, 1 Turoctocog alfa) in order to define timing and dosage of successive infusions, while 25 patients switched to EHL factors (15 Efmoroctocog alfa, 2 Ionoctocog alfa, 7 Albutrepenonacog alfa, 1 Eftrenonacog alfa). In 15 patients a population-based PK (popPK) according to WAPPS-Hemo program was also performed. The annualized bleeding rate (ABR) was counted from patient's home bleeding records for one year before PK until now. Results. According to PK data, 21 patients (75%) decreased infusion frequency (100% hemophilia B and 67% hemophilia A patients). The remaining 7 hemophilia A patients maintained the same timing in order to increase the through factor level. Notably, 1 hemophilia B patient switched from on demand treatment to prophylaxis with EHL product due to the more acceptable schedule. 66% of null mutation patients and 73% of not null mutation patients decreased timing. Of 28 patients available at follow-up, 32%, 50% and 18% decreased, increased and maintained the same annual average factor consumption/kg, respectively. All patients had a good adherence after switch. In particular, the on demand patient started a regular prophylaxis with optimal compliance. ABR displayed a reduction with a median of 0 (range 0-5) after PK analysis compared to 1 (range 0-12) before the switch. Full PK vs popPK data obtained using at least two individual PK sampling points were almost similar. Conclusions. Our results remark the necessity of PK study especially in children due to the inter-individual variability independent of genetic assessment. Regarding factor IX, PK allowed us to propose timing even longer than that recommended by prescribing indications resulting in a better personalized prophylaxis. Moreover, our study demonstrates that a full PK analysis is feasible also in children. However, given similar results, popPK could be more feasible in most patients. Regarding consumption, the reduction of only 32% of patients reflects our aim to maintain a high safety profile in an active pediatric population. Nevertheless, the mean annualized consumption was just 0.6-fold increased in the remaining patients. This approach led us to further reduce ABR and in some cases to obtain a persistent no-bleeding status even with a full active life. Disclosures No relevant conflicts of interest to declare.

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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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Retraction Of Fibrin Clots By Cultured Endothelial Cells

10.1055/s-0038-1652164 ◽

1981 ◽

Author(s):

B Barbieri ◽

G Balconi ◽

E Dejana ◽

M B Donati

Keyword(s):

Endothelial Cells ◽

Primary Cultures ◽

Fibrin Clot ◽

Test System ◽

In Vitro Test ◽

Clot Retraction ◽

System L ◽

Vitro Test ◽

Fibrin Clots

The interactions between endothelial cells (EC) and fibrin are still poorly understood. We approached this problem by studying the ability of cultured EC to induce in vitro the retraction of fibrin clots. EC were obtained from bovine aorta and human umbilical veins by collagenase treatment and grown in Eagle MEM. At the time of the test the cells were harvested from the flask by a short trypsin-EDTA treatment and resuspended in tyrode solution. The test system involved incubation of the cell suspension in a water-bath at 37°C in the presence of cell-free plasma which was clotted by thrombin. The course of retraction was followed by measuring the diameter of the clot with a microcaliper. Retraction values were expressed after calculation of percent activity by an appropriate formula. EC were found to induce the retraction of the fibrin clot to an extent which increased with the time (1-24 h) and with the number of cells in the system (l-4×l06/ml f.c.). Fibrin clot retractile (FCR) activity of EC could not be detected at 22°C or in presence of Na2-EDTA or using mechanically disrupted cells. Moreover, using batroxobin instead of thrombin as a clotting agent, no retraction occurred; FCR of EC thus showed many characteristics in common with platelet- and fibroblast- induced clot retraction.FCR activity of bovine EC increased with the number of subcultures, being very low in cells harvested from primary cultures. In contrast, human EC had high activity in primary cultures. Similarly to fibroblasts, EC with higher density in culture showed lower FCR, suggesting that con-fluency inhibits the cell contractile capacity.FCR could thus represent a simple in vitro test to further characterize the biology of EC and to evaluate their role in the development of fibrin thrombi.

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Accessibility of epitopes on fibrin clots and fibrinogen gels

Blood ◽

10.1182/blood.v77.7.1469.1469 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1469-1475 ◽

Cited By ~ 25

Author(s):

R Procyk ◽

B Kudryk ◽

S Callender ◽

B Blomback

Keyword(s):

Monoclonal Antibody ◽

Clinical Trials ◽

Ionic Strength ◽

Factor Xiii ◽

Imaging Agent ◽

Low Ionic Strength ◽

Clot Structure ◽

Radiolabeled Antibodies ◽

Fibrin Clots

Abstract Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

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