Platelet Vesicles Synergetic with Biosynthetic Cellulose Aerogels for Ultra-Fast Hemostasis and Wound Healing.

Achieving hemostasis in penetrating and irregular wounds is challenging because the hemostasis factor cannot arrive at the bleeding site, and substantial bleeding will wash away the blood clot. Since the inherently gradual nature of blood clot formation takes time, a physical barrier is needed before blood clot formation. Herein, an ultra-light and shape memory hemostatic aerogel consisting of oxidized bacterial cellulose (OBC) and platelet extracellular vesicles (pVEs) is reported. The OBC-pVEs aerogel provides a physical barrier for the bleeding site by self-expansion, absorbing the liquid from blood to concentrate platelets and clotting factors and accelerating the clot formation by activating platelets and transforming fibrinogen into fibrin. In the rat liver and tail injury models, the blood loss decreases by 73% and 59%, and the bleeding times are reduced by 55% and 62%, respectively. OBC-pVEs aerogel has also been shown to accelerate wound healing. In conclusion, this work introduces an effective tool for treating deep, non-compressible, and irregular wounds and offers valuable strategies for trauma bleeding and wound treatment.

Effects of platelets activated by different agonists on fibrin formation and thrombin generation.

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Why was the study done? Platelets and blood coagulation system interact with each other in hemostasis and intravascular thrombosis.Direct platelet effects on fibrin formation (plasma clotting), the final stage of blood coagulation cascade, have been insufficiently studied.The work is aimed at developing a method for studying platelet participation in fibrin formation in blood plasma and investigating the influence of platelet agonists on this reaction.What is new? Platelets significantly accelerate fibrin formation and their activation with various agonists (thrombin, collagen, arachidonic acid) enhances these effects.Effects of platelets on fibrin formation correlated with their ability to stimulate thrombin generation in blood plasmaEffects of platelets on fibrin formation and thrombin generation correlated with the level of phosphatidylserine exposure on their surfaceWhat is the impact? This study provides further evidence that platelet procоagulant effects on fibrin formation should be considered in investigations of platelet involvement in hemostatic and thrombotic reactions and in the evaluation of the efficacy of antiplatelet drugs.

Anti-thrombotic effects mediated by dihydromyricetin involve both platelet inhibition and endothelial protection.

Classical antithrombotics and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention. Dihydromyricetin (DHM), the most abundant flavonoid in Ampelopsis grossedentata, has unique vasoprotective effects. This study aims to characterize the antithrombotic potential of DHM. The effects of DHM on the activation of platelets and endothelial cells were evaluated in vitro. Calcium mobilization and activation of mitogen-activated protein kinases (MAPKs) were examined as the potential targets of DHM based on molecular docking analysis. The in vivo effects of DHM were determined in FeCl3-injured carotid arteries and laser-injured cremasteric arterioles. The results showed that DHM suppressed a range of platelet responses including aggregation, secretion, adhesion, spreading and integrin activation, and inhibited exocytosis, phosphatidylserine exposure and tissue factor expression in activated endothelial cells. Mechanistically, DHM attenuated thrombin-induced calcium mobilization and phosphorylation of ERK1/2 and p38 both in platelets and endothelial cells. Intravenous treatment with DHM delayed FeCl3-induced carotid arterial thrombosis. Furthermore, DHM treatment inhibited both platelet accumulation and fibrin generation in the presence or absence of eptifibatide in the laser injury-induced thrombosis model, without prolonging ex vivo plasma coagulation or tail bleeding time. DHM represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation as a result of endothelial protection.

Soluble fibrin monomer complex is associated with cardio- and cerebrovascular events in patients with heart failure.

BACKGROUND: A biomarker of fibrin formation, the soluble fibrin monomer complex (SFMC), is abnormally elevated in a variety of clinical situations of hypercoagulability. The aim of the present study was to examine the prognostic impact of SFMC, with regard to increased risk of major cardio- and cerebrovascular events (MACCE) and all-cause mortality, on patients with heart failure (HF). METHODS AND RESULTS: We conducted a prospective observational study where we analyzed data of 723 hospitalized patients with decompensated HF who were discharged alive and whose SFMC had been measured in a stable condition prior to discharge. The patients were divided into tertiles based on SFMC levels: the first (SFMC < 1.7 µg/ml, n = 250), second (≤1.8 SFMC < 2.9 µg/ml, n = 233), and third (3.0 µg/ml ≤ SFMC, n = 240) tertiles. The prevalence of chronic kidney disease and anemia was significantly higher in the third tertile than in the first and second tertiles. In contrast, age, sex, CHADS2-Vasc score, left ventricular ejection fraction, and prevalence of hypertension, diabetes and atrial fibrillation did not differ among the tertiles. In the Kaplan-Meier analysis, accumulated event rates of both MACCE and all-cause mortality progressively increased from the first to third tertiles (log-rank P < 0.05, respectively). In the multivariate Cox proportional hazard analysis, the third tertile was found to be an independent predictor of MACCE (HR 2.014, P = 0.046) and all-cause mortality (HR 1.792, P = 0.036). CONCLUSION: SFMC is an independent predictor of adverse prognosis in patients with HF.

Alterations in fibrin formation and fibrinolysis in early onset-preeclampsia: Association with disease severity.

OBJECTIVE: ; Early-onset preeclampsia is a rare pregnancy-specific disorder associated with significantly increased maternal and fetal morbidity and mortality. Whilst it is known that even normotensive pregnancies are associated with changes in clot formation and dissolution, the nature of how these changes differ in those with early onset preeclampsia has not been well established. We sought to evaluate parameters of fibrin formation and fibrinolysis in individuals with early onset preeclampsia in comparison to both pregnant and non-pregnant controls. Furthermore, such parameters were correlated with markers of disease severity in this patient cohort, including the presence of multiorgan involvement, the rate of disease progression and the extent of the anti-angiogenic state in this condition. STUDY DESIGN: ; Patients with early onset preeclampsia (N = 20) and both pregnant (N = 16) and non -pregnant (N = 16) controls were recruited from the cohort at a large urban maternity hospital which saw over 15,000 deliveries during the study period. Platelet poor plasma was prepared from collected whole blood and analysed for parameters of fibrin formation and fibrinolysis (lagtime to and rate of fibrin formation; PAI-1; PAI-2; D-dimer; plasmin-antiplasmin; tPA) in addition to markers of angiogenesis (sFLT-1; Endoglin) using commercially available specific immunoassays. RESULTS: ; The maximum rate of fibrin formation as well as PAI-1, PAI-2 and D-dimer levels were all significantly increased in those with early onset preeclampsia and pregnant controls when compared to non-pregnant controls without significant differences between the 2 former groups. Plasmin-antiplasmin levels were significantly reduced in a similar manner. tPA levels were significantly elevated in EOP compared to both pregnant and non-pregnant controls. EOP was associated with significantly increased anti-angiogenic factors (sFLT-1; Endoglin) when compared to both pregnant and non-pregnant controls. CONCLUSION: ; Markers of fibrin formation and fibrinolysis are significantly alerted in early onset preeclampsia; furthermore, certain markers correlate with disease severity in this patient cohort.

A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders.

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0·63 µg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1-deficient (-def) and α2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.

Fibrin Formation, Structure and Properties.

Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin.

"Disfibrinogenemia Jujuy", alteración asociada a trastornos hemorrágicos / "Dysfibrinogenemia Jujuy", associated to bleeding disorders / "Disfibrinogenemia Jujuy", alteração associada a distúrbios hemorrágicos

El objetivo del presente trabajo fue estudiar a una joven paciente con manifestaciones hemorrágicas, caracterizar su fibrina plasmática e identificar la posible alteración molecular del fibrinógeno de la paciente y sus familiares directos. Se diagnosticó una disfibrinogenemia en la paciente, su madre y el medio-hermano, ambos asintomáticos. En estos individuos, la formación y lisis de la fibrina plasmática difirieron de los controles. La fase lag prolongada indicó la liberación lenta y defectuosa de los fibrinopéptidos; la pendiente menor que la del control sugirió una polimerización dificultada. La DOMax inferior mostró una fibrina compuesta por fibras delgadas. La fibrinolisis inducida por estreptoquinasa resultó más rápida que la correspondiente control. La secuenciación del ADN reveló una deleción homocigota que condujo a la ausencia de la glicina 14 de la cadena Aa del fibrinógeno (AaGly14del según nomenclatura de http://www.geht.org/databaseang/fibrinogen). La madre y el medio hermano resultaron heterocigotas para la misma mutación. Esta alteración no descripta previamente, que se ha denominado fibrinógeno Jujuy, podría no interaccionar correctamente con el sitio activo de la trombina, provocando que los fibrinopéptidos se hidrolicen y liberen lentamente, originando fibras de fibrina más delgadas y degradables que las del control. Este mecanismo explicaría el sangrado moderado de la paciente.

The aim of this work was to study a young female with moderate bleeding symptoms, to characterize the plasma fibrin and to identify the possible molecular alteration in the fibrinogen of the patient and her family. A dysfibrinogenemia was diagnosed in the patient, the mother and the half-brother, both the latter asymptomatic. Kinetic parameters obtained from fibrin formation and lysis assays of the patient’s plasma samples were significantly different compared to the ones obtained with control plasma. A prolonged lag phase indicated slow and defective fibrinopeptide releases, whereas a minor slope suggested an impaired fibrin assembly. A lower ODMax revealed a fibrin network composed of thinner fibers. Fibrinolysis induced by streptokinase resulted faster than control. DNA sequencing showed a homozygous deletion leading to AaGly14del (according to http://www.geht.org/databaseang/fibrinogen). The mother and the half-brother resulted heterozygous for the same mutation. This previously undescribed alteration was named fibrinogen Jujuy. The mutate fibrinogen might not be correctly fixed to the active site of thrombin resulting in slow cleavage and release of fibrinopeptides, rendering thinner fibers, more susceptible to lysis than control. This mechanism may explain the moderate bleeding symptoms of the patient.

O objetivo do presente trabalho foi estudar uma jovem paciente com manifestações hemorrágicas, caracterizar sua fibrina plasmática e identificar a possível alteração molecular do fibrinogênio da paciente e seus familiares diretos. Foi diagnosticada uma disfibrinogenemia na paciente, sua mãe e o meio-irmão, ambos assintomáticos. Nestes indivíduos, a formação e lise da fibrina plasmática diferiram dos controles. A fase lag prolongada indicou a liberação lenta e defeituosa dos fibrinopeptídeos; a pendente menor que a do controle sugeriu uma polimerização dificultada. A DOMax inferior mostrou uma fibrina composta por fibras delgadas. A fibrinólise induzida por estreptoquinase resultou mais rápida que a correspondente controle. A sequenciação do DNA revelou uma deleção homozigótica que conduziu à ausência da glicina 14 da cadeia Aa do fibrinogênio (AaGly14del conforme nomenclatura de http://www.geht.org/databaseang/fibrinogen). A mãe e o meio irmão resultaram heterozigotos para a mesma mutação. Esta alteração não descrita previamente, que denominamos fibrinogênio Jujuy, poderia não interagir corretamente com o sítio ativo da trombina, provocando que os fibrinopeptídeos se hidrolisem e liberem lentamente, originando fibras de fibrina mais finas e degradáveis que as do controle. Este mecanismo explicaria o sangramento moderado da paciente.

Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactans from tropical green seaweeds of the order Bryopsidales.

Sulfated and pyruvylated galactans were isolated from three tropical species of the Bryopsidales, Penicillus capitatus, Udotea flabellum, and Halimeda opuntia. They represent the only important sulfated polysaccharides present in the cell walls of these highly calcified seaweeds of the suborder Halimedineae. Their structural features were studied by chemical analyses and NMR spectroscopy. Their backbone comprises 3-, 6-, and 3,6-linkages, constituted by major amounts of 3-linked 4,6-O-(1'-carboxy)ethylidene-d-galactopyranose units in part sulfated on C-2. Sulfation on C-2 was not found in galactans from other seaweeds of this order. In addition, a complex sulfation pattern, comprising also 4-, 6-, and 4,6-disulfated galactose units was found. A fraction from P. capitatus, F1, showed a moderate anticoagulant activity, evaluated by general coagulation tests and also kinetics of fibrin formation was assayed. Besides, preliminary results suggest that one of the possible mechanisms involved is direct thrombin inhibition.

Comparison by sex between thrombin generation and fibrin network characteristics in a healthy population.

BACKGROUND: The aim of the present work was to compare sex differences in thrombin generation and fibrin network characteristics in a young healthy population, and correlate thrombin generation parameters with fibrin network characteristics. METHODS: Sixty individuals aged 21 y (18-26), 50% men and 50% women were selected. Thrombin generation was performed with the Technothrombin TGA kit. Plasma fibrin formation kinetic was followed by turbidity at 350 nm, and the fibrin elastic modulus was measured with the Hemodyne. In addition, the prothrombin polymorphism G20210A was assessed. RESULTS: Thrombin generation in men was: lag time (LT): 12.5 ± 3.0 min, peak thrombin: 257 ± 135 nmol/l, and endogenous thrombin potential (ETP): 3459 ± 449 nmol/l·min, while in women the LT was shortened (9.7 ± 2.8 min, p<0.05) and thrombin formation increased (peak thrombin: 345 ± 178 nmol/l and ETP: 4017 ± 429 nmol/l·min, p<0.05). Prothrombin and fibrinogen plasma levels were similar between sexes, and the GG genotype of prothrombin G20210A polymorphism (rs1799963) had a frequency of 1. In women, fibrin formation was slightly faster and the elastic modulus was higher than men. CONCLUSIONS: The differences in thrombin generation between women and men were not related to prothrombin concentration, prothrombin polymorphism G20210A or fibrinogen concentration.

Clot waveform analysis in patients with haemophilia A.

UNLABELLED: Clot waveform analysis extends the interpretation of aPTT measurement curves. The curve is mathematically processed to obtain information about fibrin formation kinetics including semiquantitative determination of thrombin, prothrombinase and tenase activity. PATIENTS, METHOD: In this study the feasibility of clot waveform analysis for monitoring of haemophilia A was investigated using blood samples from healthy controls as well as haemophilia A patients under various clinical conditions. RESULTS: Thrombin, prothrombinase and tenase activity show a high correlation to factor VIII levels. Tenase activity was found to exhibit a linear relationship to factor VIII levels over a very large concentration range and was able to discriminate patients with severe, moderate and mild haemophilia. CONCLUSION: Clot waveform analysis is an easy, fast and cheap method to access disturbances in clot formation and can be done without any additional measurements beside an aPTT.

Thrombin generation and fibrin clot formation under hypothermic conditions: an in vitro evaluation of tissue factor initiated whole blood coagulation.

BACKGROUND: Despite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia. METHODS: Blood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography. RESULTS: Data showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. CONCLUSIONS: Induced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.

Plasma and whole blood clot strength measured by thrombelastography in patients treated with clopidogrel during acute coronary syndromes.

INTRODUCTION: Treatment with clopidogrel, a selective platelet P2Y12 receptor antagonist, reduces risk of recurrent ischemic events in patients with acute coronary syndrome (ACS), by limiting platelet aggregation and activation. Stable whole blood clot formation requires activation of platelets, generation of fibrin and final fibrin crosslinks. In this study we intended to compare plasma and whole blood thrombelastography (TEG) measurements in patients during ACS. MATERIALS AND METHODS: Whole blood and plasma samples from 32 patients with non-ST segment elevation myocardial infarction (NSTEMI) were collected after administration of clopidogrel. Whole blood and plasma fibrin clot strength (MA) were determined by TEG. Platelet aggregation was determined by light transmittance aggregometry (LTA) using adenosine 5'-diphosphate (ADP), thrombin receptor activation peptide (TRAP), or collagen as agonists. Fibrinogen and C-reactive protein (CRP) concentrations were measured by ELISA. RESULTS: Heightened plasma fibrin clot strength was associated with increased platelet reactivity stimulated by ADP (ρ=0.536; p=0.002), TRAP (ρ=0.481; p=0.007), and collagen (ρ=0.538; p=0.01). In contrast to plasma fibrin MA, whole blood MA did not correlate with platelet aggregation. Platelet count was the primary contributor to the difference in thrombin induced whole blood MA and plasma fibrin MA. Increasing levels of CRP were associated with increased plasma fibrin clot strength and platelet reactivity. CONCLUSIONS: Our data suggest that inflammation is associated with increased plasma fibrin clot strength and lower platelet inhibition by clopidogrel during ACS. Platelet count is a main contributor to additional contractile force of whole blood TEG as compared to plasma TEG during treatment with clopidogrel.

Variabilidad de las redes de fibrina / Fibrin network variability

La fibrina (polímero del fibrinógeno) es una malla proteica constituyente del tapón hemostático. Esta red se caracteriza principalmente por su estructura espacial, las dimensiones de sus fibras, el grado de ramificación, porosidad, elasticidad y rigidez, propiedades que dependen de factores como temperatura, concentración de iones y otras sustancias plasmáticas, pero principalmente de fibrinógeno, trombina y factor XIII. Cuando aumentan las concentraciones de fibrinógeno o trombina, las redes de fibrina son más densas, de menor porosidad y formadas por fibras más cortas. Las alteraciones de la molécula de fibrinógeno pueden producir fibrina anormal, de diferente disolución, ya sea que permanezca tiempos prolongados en circulación induciendo la formación de otros trombos, o bien que se degrade anticipadamente produciendo riesgo hemorrágico. El aumento de calcio incrementa la rigidez y porosidad de la red. La fibrina tiene una importante función como cofactor de la fibrinolisis. La activación del plasminógeno y la acción de la plasmina sobre la fibrina dependen de la estructura de la red. Las redes compactas son lisadas más lentamente, mientras que dentro del mismo gel, las fibras delgadas son disueltas antes que las gruesas. Por otra parte, fibras gruesas resultan mejores cofactores de la activación del plasminógeno. Conocer estos factores facilita la comprensión de situaciones fisiopatológicas que producen diferente respuesta fibrinolítica fisiológica o inducida por fármacos.

Fibrin is a protein network present in the clot. It can be characterized by its structure, fibers dimensions, ramification degree, porosity, viscoelasticity and deformability. All these properties depend on temperature, ions concentrations and on other plasmatic substances, mainly fibrinogen, thrombin and factor XIII. Greater fibrinogen or thrombin concentrations produce more dense, less porous and shorter fibered-fibrin networks. Fibrinogen alterations can generate abnormal fibrin, with different susceptibility to lysis, while calcium ions increase the fibrin networks' rigidity and porosity. Plasminogen activation and fibrinolysis rate are strongly dependent on the size and the fibrin network architecture. Tight networks are lysed more slowly while, within the same gel, thin fibers are lysed before thick ones. Otherwise, thicker fibers proved to be a better cofactor of plasminogen activation. Taking into account these factors helps understand the different fibrinolytic response, mainly when it is not explained by laboratory studies.