scholarly journals Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wai-Hoe Lau ◽  
Nathan J. White ◽  
Tsin-Wen Yeo ◽  
Russell L. Gruen ◽  
Konstantin Pervushin
Keyword(s):  
Relaxation Time ◽  
2D Nmr ◽  
Fibrin Clot ◽  
Bulk Water ◽  
Plasma Fibrinogen ◽  
Intermediate Water ◽  
Methionine Oxidation ◽  
Fibrin Polymerization ◽  
Fibrin Gels ◽  
Oxidized Fibrinogen

AbstractPlasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

scholarly journals Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

2021 ◽  
Author(s):  
Wai Hoe Lau ◽  
Nathan J. White ◽  
Tsin Wen Yeo ◽  
Russell L. Gruen ◽  
Konstantin Pervushin
Keyword(s):  
Translational Motion ◽  
Hydration Shell ◽  
Coagulation Factor ◽  
2D Nmr ◽  
Fibrin Clot ◽  
Bulk Water ◽  
Surface Relaxation ◽  
Methionine Oxidation ◽  
Fibrin Polymerization ◽  
Fibrin Gels

Abstract Plasma fibrinogen is an important coagulation factor that is susceptible to post-translational modification by oxidants. We have reported altered fibrin polymerization and increased methionine oxidation in fibrinogen after exposure to hypochlorous acid (HOCl), and similarly in the fibrinogen of severely injured trauma patients. Molecular dynamics suggests that methionine oxidation offers a mechanistic link between oxidative stress and coagulation through fibrin protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We used polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to test the hypothesis that HOCl oxidation alters fibrinogen conformation in the prefibrillar state and T2 water surface relaxation of fibrin fiber assemblies. We found that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma disrupted fibrin polymerization similarly to competitive inhibition of polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of 1H fibrinogen at 25oC demonstrated that fibrinogen oxidation increased translational diffusion coefficient by 17.4%, suggesting a more compact and rapidly translational motion of the protein with oxidation. 2D-NMR analysis of control plasma fibrin gels indicated that water existed in two states, namely intermediate (T2i) in the hydration shell of fibrin fibers, and bulk (T2) within the gel. T2 relaxation of bulk water protons was decreased 2-fold in oxidized fibrin gels and was inversely proportional to gel fiber density (T2). The fast exchange of water protons between hydration shell (T2i) and bulk water, indicating oxidation increased fiber hydration and formed densely packed fibrin gels. We have confirmed experimentally that HOCl oxidation affected native fibrinogen and fibrin gel structures and have demonstrated that NMR can serve as a valuable tool to probe the oxidative rearrangement of fibrin clot structure.

Fibrinogen α-Chain Resistance to Arvin (Ancrod) in DIC

1975 ◽  
Author(s):  
R. S. Lane ◽  
O. H. Baugh ◽  
P. T. Flude
Keyword(s):  
Molecular Weight ◽  
Aortic Dissection ◽  
Dissecting Aneurysm ◽  
Plasma Fibrinogen ◽  
Low Molecular Weight ◽  
Fibrin Polymerization ◽  
Rapid Isolation ◽  
Α Chain ◽  
High Concentrations ◽  
Marked Resistance

In high concentrations, Arvin (Ancrod) splits fibrinogen α-chains into fragments of 39,000 and 31,000 molecular weight. In a patient with mild chronic DIC, secondary to a dissecting aneurysm, plasma fibrinogen exhibited marked resistance to normal α-chain proteolysis by Arvin. The patient showed a moderate haemostatic defect acquired from the time of aortic dissection: bleeding was stopped with EACA. Tests were consistent with the presence in plasma of high and low molecular weight FDP, thus deficient haemostasis could be explained by defective fibrin polymerization and X-linking.The α-chain resistance to Arvin was a coincidental finding. Does this behaviour represent a defect and, if so, is it congenital, or acquired representing a previously unencountered phenomenon associated with DIG? The data show Arvin to be a valuable agent for the rapid isolation and investigation of fibrinogen in normal and pathological plasmas.

scholarly journals Differential Sialic Acid Content in Adult and Neonatal Fibrinogen Mediate Differences in Clot Polymerization

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Kimberly A Nellenbach ◽  
Nina A Guzzetta ◽  
Ashley C Brown
Keyword(s):  
Sialic Acid ◽  
Acid Concentration ◽  
Acid Content ◽  
Blood Clot ◽  
Enzymatic Digestion ◽  
Fibrin Clot ◽  
Blood Products ◽  
Sialic Acid Content ◽  
Fibrin Polymerization ◽  
Fibrin Clots

Recent studies have identified several major qualitative and quantitative differences in important hemostatic proteins between adult and neonatal humans, including the primary coagulation protein fibrinogen.Despite these differences, neonates with post-operative bleeding from procedures requiring cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) are treated with transfusions of adult blood products, namely adult fibrinogen. The effectiveness of such transfusions is inconsistent in neonates and often results in a deficient fibrin matrix structure which may not be sufficient for mitigating bleeding. Our recent studies have also identified differences at the bulk clot level between purified neonatal and adult fibrin clots, including major structural and functional distinctions, which could contribute to these outcomes. Notably, adult fibrinogen degrades slower than neonatal clots, therefore transfusion of adult blood products to neonatal patients could contribute to thrombotic complications. Given the inconsistent results and potential complications from the transfusion of adult blood products to neonatal patients, there is a critical need to better understand the mechanistic differences in hemostatic processes between adults and neonates. Neonates possess a molecular variant of fibrinogen known as fetal fibrinogen. Increased sialic acid concentration compared to adults has been identified in many neonatal glycoproteins across physiological systems, including fetal fibrinogen. Studies have shown that fibrin clot properties are influenced by fibrin polymerization mechanisms and post translational modifications (e.g. sialic acid). For example, an increased sialic acid content in the dysfibrinogenemia associated with liver disease has been associated with an altered fibrin clot structure. Additionally, recent work from our group has found that the increased sialic acid in neonatal fibrin networks results in significantly greater fibroblast attachment than adult networks. Therefore, we hypothesized that differences in neonatal and adult fibrin clot properties are due to mechanistic differences in fibrin polymerization between neonates and adults owing to altered sialic acid concentrations. The activation of fibrinogen and conversion into fibrin by the proteolytic enzyme thrombin is essential for the formation of a stable blood clot and the cessation of bleeding. Thrombin converts soluble fibrinogen to insoluble fibrin via cleavage of fibrinopeptides A and B, exposing fibrin knobs A and B. Fibrin protofibrils are then formed from the noncovalent binding of fibrin knobs to complementary fibrin holes a and b on adjacent proteins. In adults, fibrin A:a knob:hole interactions are critical for polymerization. However, these mechanisms have not been explored in neonates. Therefore, we characterized the influence of sialic acid on the knob:hole interactions in neonatal fibrin polymerization. We first investigated the influence of sialic acid concentration on neonatal fibrin polymerization by removing sialic acid via enzymatic digestion and performing structural and functional analysis on desialylated fibrinogen. Desialylated adult and neonatal fibrinogen had roughly equivalent structure, polymerization kinetics, and clottability results. These results indicate that differential sialylation may at least partially explain functional differences been adult and neonatal clots. Additionally, we investigated the role that sialylation plays on neonatal fibrin polymerization dynamics by comparing neonatal or adult fibrin clots formed with snake venom thrombin-like enzymes (svTLE) that preferentially cleave either A or B fibrinogen fibrinopeptides or thrombin. Structural, mechanical, fibrinolytic, and polymerization assays were conducted. Quantitative release of fibrinopeptides was determined via ELISA. Results indicate neonatal fibrin polymerization mechanisms are more dependent on B:b knob:hole interactions than adult fibrin. Results from this study provide insight into the mechanism of the neonatal clotting process and are a critical contribution for the development of neonatal-specific treatments for bleeding and thrombosis. Disclosures Brown: Selsym Biotech, Inc.: Other: Founder and CEO.

Hyaluronan stimulates tumor cell migration by modulating the fibrin fiber architecture

1999 ◽  
Vol 112 (13) ◽  
pp. 2241-2251 ◽  
Author(s):  
W. Hayen ◽  
M. Goebeler ◽  
S. Kumar ◽  
R. Riessen ◽  
V. Nehls
Keyword(s):  
Cell Migration ◽  
Tumor Cell ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Boyden Chamber ◽  
Fibrin Polymerization ◽  
Fibrin Gels ◽  
Tumor Cell Migration ◽  
And Migration

The glycosaminoglycan hyaluronan, which supports tumor cell migration and metastasis, interferes with fibrin polymerization and leads to increased fiber size and porosity of fibrin clots. Here we have studied the proportionate effect of fibrin polymerization on hyaluronan-mediated migration of glioblastoma cells. The structural and physical properties of hyaluronan-containing fibrin gels were analyzed by turbidity measurement, laser scanning microscopy, compaction assay, and calculation of pore size by liquid permeation. When fibrin polymerized in the presence of hyaluronan or dextran, the resulting gels strongly stimulated cell migration, and migration significantly correlated with fiber mass-to-length ratios and pore diameters. In contrast, cell migration was not induced by addition of hyaluronan to supernatants of already polymerized gels. Hyaluronan-mediated migration was inhibited in fibrin gels by antibodies to alphav- and beta1integrins and the disintegrin echistatin, but not by antibodies to the hyaluronan receptor CD44 (up to 50 microg/ml). As a control, we show that anti-CD44 (10 microg/ml) inhibited cell migration on a pure hyaluronan matrix using a two-dimensional Boyden chamber system. In contrast to three-dimensional migration, the migration of cells on the surfaces of variably structured fibrin gels was not significantly different, indicating that increased gel permeability (porosity) may account for hyaluronan-mediated migration. We conclude that, in complex three-dimensional substrates, the predominant effect of hyaluronan on cell migration might be indirect and requires modulation of fibrin polymerization.

Phenotype and Genotype of 22 Subjects with Heritable Dysfibrinogenaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1212-1212
Author(s):  
Katharine Hodby ◽  
Emma Phillips ◽  
Suzi Shapiro ◽  
Michael Laffan ◽  
Richard Manning ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Family Members ◽  
Plasma Fibrinogen ◽  
Case Report Form ◽  
Fibrinogen Concentration ◽  
Fibrin Polymerization ◽  
Exon 2 ◽  
Consistent Finding ◽  
Clinical Records ◽  
Index Cases

Abstract Abstract 1212 The heritable dysfibrinogenaemias (HD) are a heterogenous group of qualitative disorders of fibrinogen in which there is abnormal fibrin clot formation. Affected individuals usually display prolonged thrombin (TCT) and reptilase clotting times (RCT) and reduced plasma fibrinogen activity. The plasma fibrinogen concentration is normal or is reduced less than the fibrinogen activity. HD is associated with pathogenic mutations in FGA, FGB or FGG genes that encode the fibrinogen Aα, Bβ and g chains respectively. However, most clinical descriptions of HD comprise single case reports and the range of phenotype and genotype in the wider population is difficult to estimate. In order to improve understanding of this disorder, we now report a cross-sectional survey of 22 subjects with HD identified at 2 UK regional Haemostasis centres from 2001–11. We recruited index cases from our Haemostasis laboratory referral practice who satisfied all the following criteria- 1) prolonged TCT; 2) prolonged TCT with protamine (TCTP) and/or prolonged RCT; 3) reduced Clauss fibrinogen and 4) normal fibrinogen dry clot weight. Demographic and clinical characteristics were recorded using a standardised case report form. Genetic analysis was performed by Sanger sequencing of FGA exon 2, FGB exon 2 and FGG exon 8 which we identified as mutation hotspots from the fibrinogen mutation database (http://www.geht.org/databaseang/fibrinogen/). Phenotype and genotype was also determined in available first degree family members. We identified 16 index cases and 6 family members with HD (median age 41, range 12–78; 9 males). HD was identified in plasma submitted to our Haemostasis laboratories for the investigation of bleeding in 8/16 (50%) index cases, for stroke in 1/16 (6%) and for ‘routine coagulation screening’ in 7/16 (44%). Mild mucocutaneous bleeding or menorrhagia was documented in the clinical records of 13/22 (59%) subjects and thrombotic stroke in 1/22 (5%). 8/22 (36%) subjects were asymptomatic. The prothrombin time (PT) was prolonged in 21/22 (95%) subjects (median 15.3 s; range 11.4–16.7; reference interval (RI) 9.6–11.6) but the activated partial thromboplastin time (aPTT) was prolonged in only 1/22 (5%; median 23.8 s; range 22.8–37.0, RI 24.0–32.0). The median TCT was 28.6 s (range 20.6–45.7; RI 13–19) and TCTP 31.9 s (n=11; range 20.0–35.5; RI 13–19). The median RCT was 37s (n=12; range 19–68; RI 15–19), Clauss fibrinogen 0.49 g/dL (range 0.29–1.3; RI 1.8–3.6) and dry clot weight 2.3 g/dL (range 1.9–4.2; RI 1.8–3.6). Heterozygous missense nucleotide variations were identified in the candidate exons of the fibrinogen genes in all 16/16 (100%) index cases and segregated with the HD phenotype in all available family members. These variations predicted aminoacid substitutions at the fibrinopeptide A cleavage site in the fibrinogen Aa chain in 10/16 (63%) index cases (FGA p.R35H (n=4); p.R35S (n=3); p.R35P (n=2) and p.G36S (n=1); numbered from RefSeq NP_000499.1) or at nearby residues (FGA p.V39D (n=1) and p.F27C (n=1)). FGG variations were identified in 3 index cases that predicted aminoacid substitutions in the fibrinogen g chain region necessary for D:D domain interactions during fibrin monomer assembly (FGG p.R301H (n=1), p.D342G (n=1) and p.A367T (n=1); RefSeq NP_000499.1). One subject harboured a FGB variation that predicted a p.R44C substitution (Refseq NP_005132.2) at the fibrinopeptide B cleavage site in the fibrinogen Bb chain. The FGA p.R35S, p.R35H, FGB p.R44C and FGG p.R301H and p.A367T substitutions have been associated previously with HD. We show that the HD laboratory phenotype is rare and was frequently identified in subjects with mild bleeding. However, HD was also commonly asymptomatic and did not segregate with bleeding symptoms within affected families suggesting that the HD variants identified in this study do not confer significant bleeding risk. The prevalence of mild bleeding in some study subjects may reflect recruitment bias. We also show that HD is strongly associated with nucleotide variations in the FGA, FGB or FGG genes. All the observed nucleotide variations have been previously associated with HD and/or predict aminoacid substitutions in critical regions for normal fibrin polymerization. Defective fibrin polymerization may affect endpoint determination in some automated clotting assays and is likely to account for the consistent finding of prolonged PT in the study subjects. Disclosures: No relevant conflicts of interest to declare.

A study of pine resin in softwood by 1D and 2D time-domain NMR

Holzforschung ◽  
2020 ◽  
Vol 74 (9) ◽  
pp. 839-852
Author(s):  
Gao Xin ◽  
Zhou Fan ◽  
Fu Zongying ◽  
Zhou Yongdong
Keyword(s):  
Relaxation Time ◽  
Time Domain ◽  
Bound Water ◽  
Quantitative Relationship ◽  
2D Nmr ◽  
Radiata Pine ◽  
Sample Treatment ◽  
Isothermal Adsorption ◽  
Pine Resin ◽  
Nmr Signals

AbstractTime-domain nuclear magnetic resonance (TD-NMR) is widely used in the investigation of wood-water relationship. However, some ambiguities between the NMR signals and the components in wood remain unresolved, particularly the effect of pine resin on NMR signals. To clarify these ambiguities and increase the use of TD-NMR in wood research, different sample treatment methods were studied, including air-drying, low-temperature vacuum-drying, diethyl ether extraction and moisture isothermal adsorption. The corresponding one-dimensional (1D) T1, T2 and two-dimensional (2D) T1-T2 correlation relaxation time distributions of radiata pine and Douglas fir samples were investigated. The NMR signals accounted for “longer relaxation-time components” below the fiber saturation point (FSP), but overlaped in parts of the 1D relaxation time distributions making it difficult to distinguish between pine resin and moisture. The 2D T1-T2 correlation relaxation time distributions produced a better distinction between pine resin and bound water. This distinction established a quantitative relationship between pine resin, moisture and 2D NMR signal amplitudes.

scholarly journals A heparinoid from peony roots (Paeonia anomala): effects on polymerization and dissolution of fibrin in thrombosis

2020 ◽  
pp. 80-84
Author(s):  
М.С. Успенская ◽  
М.Г. Ляпина ◽  
Е.С. Майстренко
Keyword(s):  
Ex Vivo ◽  
Coagulation Factor ◽  
Fibrin Clot ◽  
Rat Plasma ◽  
Male Rats ◽  
Factor Xiiia ◽  
Thrombin Time ◽  
Fibrin Polymerization ◽  
Thrombin Activity ◽  
Induction Experiment

Исследование гепаринов и гепариноидов в качестве антитромботических агентов актуально для физиологии и медицины. Многие растения включают гепариноподобные компоненты (гепариноиды), которые препятствуют тромбообразованию. Цель - установление влияния экстракта из корней пиона «Марьин корень» (Paeonia anomala), содержащего гепариноид, на полимеризацию фибрина при процессах тромбообразования ex vivo и определение возможных механизмов его антитромботического действия. Методика. Исследовано влияние гепариноида из пиона (Paeonia anomala) на процессы растворения фибрина в условиях тромбообразования ex vivo. Разработана модель тромбоза (МТ) ex vivo. К плазме крови крыс (объем 0,2 мл) добавляли 2 NIH ед. тромбина (0,05 мл), фибриновый сгусток образовывался в течение 2-3 мин. Экстракт гепариноида из пиона (0,1 мл 0.5%-й) добавляли к предобразованному сгустку через 12 мин после моделирования тромбоза (опыт А), или одновременно с добавлением тромбина к плазме крови (опыт Б). Использовали венозную (из v. jugularis) кровь крыс-самцов Wistar. Полимеризацию фибрина выявляли по тесту фибриндеполимеризационной активности (ФДПА) плазмы крови на нестабилизированном фибрине. В продуктах растворения фибрина под влиянием гепариноида оценивали активность тромбина (по тесту тромбинового времени), cвертывающего фактора XIIIa (по определению активности фактора XIIIa) и ФДПА. Результаты. В опыте А спустя 10 мин после добавления гепариноида к предобразованному сгустку отмечалось появление в нем жидкой фазы, что свидетельствовало о способности исследуемого гепариноида растворять фибрин. В опыте Б сгусток или не образовывался, или же был рыхлым. Полученные данные свидетельствовали об ингибировании процесса полимеризации фибрина под влиянием гепариноида. Выявлены антитромбиновые и антифибринстабилизирующие эффекты гепариноида из пиона при добавлении к фибриновому сгустку. Рассматриваются возможные механизмы действия гепариноида на блокаду полимеризации фибрина. Заключение. Растительный гепариноид препятствовал процессам полимеризации фибрина или растворял образующиеся фибриновые сгустки, что связано с его антитромбиновым и антифибринстабилизирующим действием. Studying heparins and heparinoids as antithrombotic agents is relevant for physiology and medicine. Many plants contain heparin-like components (heparinoids) that prevent thrombosis. The aim of the study was to identify effects of a heparinoid obtained from peony (Maryin root, P. anomala) roots on polymerization of fibrin and fibrinolytic activity of blood plasma and to suggest possible mechanisms of these effects in experimentally induced ex vivo thrombosis in rats. Methods. The effect of peony (Paeonia anomala) root heparinoid on fibrin dissolution was studied in the ex vivo conditions of thrombosis. For ex vivo modeling of thrombosis (MT), 2 NIH units of thrombin (0.05 ml) were added to 0.2 ml of rat plasma. A fibrin clot formed within 2-3 minutes. The peony heparinoid extract (0.5%, 0.1 ml) was added either to the pre-formed clot at 12 min after MT induction (experiment A) or simultaneously with the addition of thrombin to plasma (experiment B). Jugular vein blood from Wistar male rats was used. Fibrin polymerization was detected using a plasma fibrin-depolymerization activity (FDPA) test on non-stabilized fibrin. Thrombin activity (thrombin time test), coagulation factor XIIIa activity, and FDPA were evaluated in products of fibrin dissolution induced by the heparinoid. Results. In experiment A, at 10 min after the addition of heparinoid to the pre-formed clot, a liquid phase emerged, which indicated an ability of the pion heparinoid to dissolve fibrin. In experiment B, the clot either did not form or was liquid. These results indicated inhibition of fibrin polymerization under the action of the heparinoid. Therefore, the peony heparinoid added to the fibrin clot antagonized thrombin and fibrin stabilization. The article addresses possible mechanisms of the heparinoid inhibition of fibrin polymerization. Conclusion. The studied plant heparinoid prevented processes of fibrin polymerization or dissolved formed fibrin clots due to depression of thrombin activity and fibrin stabilization.

scholarly journals Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

2021 ◽  
Author(s):  
Kimberly Nellenbach ◽  
Alexander Kyu ◽  
Nina Guzzetta ◽  
Ashley Brown
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Fibrin Clot ◽  
Fibrinopeptide A ◽  
Sialic Acid Content ◽  
Fibrin Polymerization ◽  
Fibrin Networks ◽  
Molecular Variant ◽  
Mixed Efficacy ◽  
And Function

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared to adults. Despite these differences, adult fibrinogen is used for treatment of bleeding in neonates, with mixed efficacy. In order to determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compare to those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin like enzymes (SVTLEs) that preferentially cleave fibrinopeptide A or B. We determined that sialic acid content of neonatal fibrinogen is a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal specific treatments of bleeding.

scholarly journals A Universal Heparin Antidote with Negligible Effect on Fibrin(ogen) and Plasma Coagulation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4231-4231
Author(s):  
Manu Thomas Kalathottukaren ◽  
Rajesh A Shenoi ◽  
Lai FL Benjamin ◽  
Fred Rosell ◽  
Jayachandran N Kizhakkedathu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Lag Time ◽  
Fibrin Clot ◽  
Coagulation System ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Fibrin Polymerization ◽  
Maximum Absorbance ◽  
Fiber Size ◽  
Clot Structure

Abstract Background and Objective Anticoagulants play a pivotal role in the treatment of thromboembolic disorders. Haemorrhage in surgical patients receiving anticoagulants is a major concern. Antidotes are administered to counteract anticoagulation and to restore normal hemostasis. To date, protamine sulphate (PS), a cationic polypeptide is the only clinically approved antidote for unfractionated heparin. PS has toxic side effects and limitations. Inability of PS to completely reverse low molecular weight heparins and fondaparinux is due to its low binding affinity to these drugs. However, PS interacts with coagulation proteins such as fibrinogen to form aggregates which leads to cardiovascular adverse effects. Recently, we developed a synthetic universal heparin reversal agent (UHRA) with high binding affinity to heparins. In vivo studies revealed that UHRA completely reverse the activity of all clinical available parenteral anticoagulants and is nontoxic. This study aims to demonstrate the nontoxic nature of UHRA by assessing its influence on fibrinogen, fibrin clot architecture, plasma clotting and clot lysis. Methods UHRA was developed by incorporating tertiary amine based heparin binding groups on a dendritic hyperbranched polyglycerol scaffold and capping it with methoxy polyethylene glycol chains. Recalcification and tissue factor (TF) initiated turbidimetric plasma clotting assays was performed to understand the impact of UHRA on coagulation system. The interaction of UHRA on fibrinogen was investigated by fibrinogen aggregation assay, fibrin polymerization assay and by spectroscopic analysis (fluorescence and circular dichroism (CD)). The influence of UHRA on fibrin clot architecture was evaluated by scanning electron microscopy (SEM).The anticoagulant neutralization (heparins) by UHRA was studied by fluorogenic thrombin generation assay (TGA) in human platelet-rich plasma (PRP). The lysis of TF-induced plasma clot containing UHRA or PS exposed to exogenous tissue plasminogen activator (t-PA) was studied by turbidimetric assay. Results and discussion Results from the plasma clotting assays showed that UHRA did not alter the clotting parameters compared to PS (TF initiated lag time and maximum absorbance, control vs UHRA 200 mcg/mL, p=0.21 and 0.16, respectively; lag time and maximum absorbance in recalcification, control vs UHRA 200mcg/mL, p=0.08 and 0.13, respectively) suggesting that UHRA has no effect on coagulation system at the concentration studied (Figure 1). Unlike protamine, the fibrinogen aggregation and fibrin polymerization assay was not influenced by UHRA over a broad range of concentrations from 0.05mg/mL to 1mg/mL. Together with tryptophan fluorescence quenching measurements (Figure 2) and fibrinogen secondary structure measurements corroborates that UHRA is not interacting with fibrinogen. The results are quite different from PS and other synthetic cationic polymers which interact with fibrinogen eliciting aggregation and conformational changes. Fibrin clots generated in presence of UHRA (even at 0.5 mg/mL) showed similar structure and fiber size remains same as normal fibrin clot (control vs UHRA 0.5 mg/mL clot, p= 0.12) (Figure 3). On the other hand, fibrin clots formed in the presence of 0.05mg/mL PS (clinical dose) increased the fiber size and changed the clot structure dramatically (control vs PS 0.05mg/mL clot, p< 0.0001). Our plasma clot lysis studies in the presence of exogenous t-PA demonstrate that UHRA did not enhance clot degradation unlike protamine. UHRA restored thrombin levels in anticoagulated PRP (heparinized) demonstrating the efficacy. Conclusion and significance Our studies demonstrate that universal heparin antidote, UHRA, has negligible impact on fibrinogen, fibrin polymerization, clot structure, clot degradation and the coagulation system revealing their excellent hemocompatibility compared to protamine. Our results support the fact that UHRA could be an ideal antidote to restore hemostasis following invasive surgical procedures and to address bleeding complications by heparin based anticoagulants. Figure 1 Figure 1. Figure 3 Figure 3. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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