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

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.

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

Scientific Reports ◽

10.1038/s41598-021-94401-3 ◽

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.

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

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/0031-2991.2020.02.80-84 ◽

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.

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NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure–Activity Relationships

International Journal of Molecular Sciences ◽

10.3390/ijms22020880 ◽

2021 ◽

Vol 22 (2) ◽

pp. 880

Author(s):

Thomas Schmitz ◽

Ajay Abisheck Paul George ◽

Britta Nubbemeyer ◽

Charlotte A. Bäuml ◽

Torsten Steinmetzer ◽

...

Keyword(s):

Structural Information ◽

Coagulation Factor ◽

Md Simulations ◽

2D Nmr ◽

Label Free ◽

2D Nmr Spectroscopy ◽

Structure Information ◽

Structure Activity Relationships ◽

Structure Activity ◽

Blood Sucking

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1–Cys37) and the flexible C-terminal part (Arg38–Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure–activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

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Factor XIII and Fibrin Clot Properties in Acute Venous Thromboembolism

International Journal of Molecular Sciences ◽

10.3390/ijms22041607 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1607

Author(s):

Michał Ząbczyk ◽

Joanna Natorska ◽

Anetta Undas

Keyword(s):

Venous Thromboembolism ◽

Factor Xiii ◽

Active Form ◽

Coagulation Factor ◽

Proteome Analysis ◽

Fibrin Clot ◽

Subsequent Increase ◽

Vein Thrombosis ◽

Acute Pe ◽

Fxiii Activity

Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII’s involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.

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FACTOR XIII CONCENTRATE FOR PROPHYLAXIS OF REBLEEDING IN SUBARACHNOID HEMORRHAGE (SAH) - RESULTS OF A PROSPER TIVE MULTICENTER PILOT STUDY

10.1055/s-0038-1643312 ◽

1987 ◽

Author(s):

A Thie ◽

T h Henze ◽

D Deggar ◽

M Obering ◽

R Clemers ◽

...

Keyword(s):

Subarachnoid Hemorrhage ◽

Pilot Study ◽

Mortality Rate ◽

Factor Xiii ◽

Coagulation Factor ◽

Fibrin Clot ◽

Stage I ◽

Stage Iii ◽

Double Blind Study ◽

Overall Mortality

Rebleeding occurs in subarachnoid hemorrhage (SAH) in 20 - 25 % of patients, with a mortality rate being above 50 %. The cause of rebleeding is considered to be premature fibrinolysis of the fibrin clot surrounding the site of rupture. Since the stability of the fibrin clot is influenced by the activity of coagulation factor XIII, and moreover, a factor XIII deficiency has been reported in SAH patients, the question arises as to whether the incidence of rebleeding can be influenced by the administration of F XIII concentrate.During a period of 6 months, 69 patients with acute SAH were enlisted in an open, prospective, multicenter study. On admission, 5 patients were classified as stage I (7.2%) according to the Hunt and Hess scale, 22 as stage III (31.9%), 11 as stage IV (16%) and 9 as stage V (13%). Aneurysm was confirmed by angiography in 52 patients (75%). All the patients received 10 x 1250 U F XIII concentrate during the first 15 days after the initial hemorrhage. Surgery on the aneurysm was performed between day 3 and 32 (median: day 13) in 35 patients.A total of 7 rebleedings occurred in 6 patients (8.7%), of whom 2 were stage I - II and 4 were stage III - V cases. Cerebral infarction was observed in 10 patients (14.5%), and hydrocephalus requiring shunting occurred in 1 patient. There were no cases of peripheral thromboses or embolisms. After 4 weeks, the overall mortality rate was 26%. (stage I - II: 11.1%, stage III - V: 37.5%).The conventional approach in the prophylaxis of rebleeding in SAH is an early operation or intravenous administration of antifibrinolytics. However, as none of these measures significantly reduce overall mortality, the present pilot study investigated a new, therapeutic approach in which F XIII concentrates were administered in order to stabilize the fibrin clots and prevent premature fibrinolysis. The data so far show that Fibrogammin P is an effective and well tolerated agent for the prophylaxis of post-SAH rebleeding. In order to statistically confirm the results of the pilot study, we have, in the meantime, started a prospective, randomized, placebo-controlled, multicenter double-blind study, which will involve 750 patients over a period of 2 years.

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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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Plasmin-Mediated Coagulation Factor Xa Derivative, Xa33/13, Enhances Plasminogen Activation and Fibrin Clot Lysis.

Blood ◽

10.1182/blood.v108.11.1717.1717 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1717-1717

Author(s):

Ed L.G. Pryzdial ◽

Kimberley Talbot ◽

Scott C. Meixner

Keyword(s):

Factor Xa ◽

Coagulation Factor ◽

Fibrin Clot ◽

Clot Formation ◽

Clot Lysis ◽

Fragmentation Pattern ◽

Plasmin Activity ◽

Anionic Phospholipid ◽

Western Blots ◽

Chromogenic Assay

Abstract Previous reports showed that coagulation factor Xa (FXa) treated with the fibrinolysis protease, plasmin, loses procoagulant activity and can accelerate tissue plasminogen activator (tPA). The resulting FXa derivative, Xa33/13, acquires binding sites for plasminogen and was consequently presumed to be the form of FXa involved in generating plasmin. By evaluating the function of purified Xa33/13, the current work addresses the hypothesis that conversion of FXa to Xa33/13 is the basis of enhanced plasmin generation by tPA and that this newly acquired FXa function can also contribute to clot lysis. Using purified proteins in a chromogenic assay, both Xa33/13 and FXa were found to increase the generation of plasmin activity by tPA at least 10-fold. To explain the involvement of FXa reported previously, Western blots demonstrated a correlation between the disappearance of FXa, appearance of Xa33 antigen and enhanced plasmin production. The Western blots also revealed that detection of Xa33 antigen is lost at later time points, which explains the observation that plasmin generation is eventually shut down during the chromogenic assay. Despite fibrin being recognized as the principal accelerator of tPA, at concentrations as low as 10 nM, either Xa33/13 or FXa reduced the lysis time of thrombin-mediated fibrin clot in a turbidity assay. The Xa33/13 or FXa could be added during clot formation before the tPA or after clot formation with the tPA to facilitate a 50–70% reduction in the time required to achieve half maximal lysis at 0.1 μM. The time-dependent formation of Xa33 antigen in plasma that was induced to clot with tissue factor, anionic phospholipid vesicles and Ca2+, demonstrated that the plasmin-mediated fragmentation pattern of purified FXa represents the complex physiological mixture. However, the use of specific inhibitors and immuno-depleted plasma indicated that plasmin is not required for production of Xa33 antigen in plasma. Cumulatively the data presented here support a role for coagulation FXa in fibrinolysis. These diametrically opposing functions derived from the same molecule, may contribute to the balance between coagulation and fibrinolysis, thereby facilitating tolerance for a wide normal physiological range of FX and possibly other coagulation proteins.

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

Blood ◽

10.1182/blood-2020-139965 ◽

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.

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Normalization of Blood Clotting Characteristics Using Prothrombin Complex Concentrate, Fibrinogen and Fxiii In an Albumin Based Fluid: Experimental Studies In Thromboelastometry.

10.21203/rs.3.rs-98928/v1 ◽

2020 ◽

Author(s):

Tobias Koller ◽

Nadia Kinast ◽

Andres Guilarte Castellanos ◽

Sergio Perez Garcia ◽

Pilar Paniagua Iglesias ◽

...

Keyword(s):

Whole Blood ◽

Factor Xiii ◽

Prothrombin Complex Concentrate ◽

Massive Transfusion ◽

Experimental Studies ◽

Coagulation Factor ◽

Fibrin Clot ◽

Fibrinogen Concentrate ◽

Coagulation Factor Concentrates

Abstract Background: Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with capability to restore coagulation could be a desirable future treatment component in massive transfusion.Methods: Starting from a coagulation factor and blood cell free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under presence of platelets was performed for comparability to whole blood conditions.Results: Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 minutes and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 minutes and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood.Conclusions: Combinations of coagulation factor concentrates suspended in albumin solutions have the capacity to restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation-restoring characteristics might offer new treatment alternatives for massive transfusion.Trial registration: Study registered at the institutional ethic committee “Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165.

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Normalization Of Blood Clotting Characteristics Using Prothrombin Complex Concentrate, Fibrinogen And FXIII In An Albumin Based Fluid: Experimental Studies In Thromboelastometry.

10.21203/rs.3.rs-98928/v2 ◽

2021 ◽

Author(s):

Tobias Koller ◽

Nadia Kinast ◽

Andres Guilarte Castellanos ◽

Sergio Perez Garcia ◽

Pilar Paniagua Iglesias ◽

...

Keyword(s):

Whole Blood ◽

Factor Xiii ◽

Prothrombin Complex Concentrate ◽

Massive Transfusion ◽

Experimental Studies ◽

Coagulation Factor ◽

Fibrin Clot ◽

Fibrinogen Concentrate ◽

Coagulation Factor Concentrates

Abstract Background: Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with the capability to restore coagulation could be a desirable future treatment component in massive transfusion.Methods: Starting from a coagulation factor and blood cell-free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under the presence of platelets was performed for comparability to whole blood conditions.Results: Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 minutes and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 minutes and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood.Conclusions: Combinations of coagulation factor concentrates suspended in albumin solutions can restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation-restoring characteristics might offer new treatment alternatives for massive transfusion.Trial registration: Study registered at the institutional ethic committee “Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165.

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