Plant-derived compounds normalize platelet bioenergetics and function in hyperglycemia.

Background: Polyphenols have been shown to decrease oxidative stress and modulate glycemic response. Nevertheless, their effect on platelet bioenergetics and clot structure in diabetes and hyperglycemia is unknown. Objectives: To investigate the effect of polyphenols on human platelet bioenergetics and its subsequent effect on clot structure in normoglycemia vs acute hyperglycemia in vitro. Methods: Four polyphenols (resveratrol, hesperetin, epigallocatechin gallate [EGCG], and quercetin) were selected for initial analysis. Healthy volunteers' isolated platelets/platelet-rich plasma were treated with 5 or 25 mM glucose to represent normoglycemia and acute hyperglycemia, respectively. Platelet-derived reactive oxygen species (ROS), citrate synthase activity (mitochondrial density), mitochondrial calcium flux, and mitochondrial respiration were performed following exposure to polyphenols (20 µM, 1 hour) to determine their effects on platelet bioenergetics. Procoagulant platelets (annexin V) and fibrin fiber density (Alexa Fluor-488 fibrinogen; Invitrogen) were analyzed by laser scanning confocal microscopy, while clot porosity was determined using platelet-rich plasma following exposure to polyphenols (20 µM, 20 minutes). Results: Acute hyperglycemia increased ROS, mitochondrial calcium flux, maximal respiration, and procoagulant platelet number. Resveratrol, quercetin, and EGCG reduced platelet ROS in normoglycemic and acute hyperglycemic conditions. Mitochondrial density was decreased by quercetin and EGCG in normoglycemia. Resveratrol and EGCG reduced mitochondrial calcium flux in acute hyperglycemia. Resveratrol also decreased procoagulant platelet number and attenuated oxygen consumption rate in normoglycemia and acute hyperglycemia. No effect of hyperglycemia or polyphenols was observed on fibrin fiber density or clot pore size. Conclusion: Our results suggest polyphenols attenuate increased platelet activity stemming from hyperglycemia and may benefit thrombosis-preventative strategies in patients with diabetes.

Ischaemic Stroke, Thromboembolism and Clot Structure.

Ischaemic stroke is a major cause of morbidity and mortality worldwide. Blood clotting and thromboembolism play a central role in the pathogenesis of ischaemic stroke. An increasing number of recent studies indicate changes in blood clot structure and composition in patients with ischaemic stroke. In this review, we aim to summarise and discuss clot structure, function and composition in ischaemic stroke, including its relationships with clinical diagnosis and treatment options such as thrombolysis and thrombectomy. Studies are summarised in which clot structure and composition is analysed both in vitro from patients' plasma samples and ex vivo in thrombi obtained through interventional catheter-mediated thrombectomy. Mechanisms that drive clot composition and architecture such as neutrophil extracellular traps and clot contraction are also discussed. We find that, while in vitro clot structure in plasma samples from ischaemic stroke patients are consistently altered, showing denser clots that are more resistant to fibrinolysis, current data on the composition and architecture of ex vivo clots obtained by thrombectomy are more variable. With the potential of advances in technologies underpinning both the imaging and retrieving of clots, we expect that future studies in this area will generate new data that is of interest for the diagnosis, optimal treatment strategies and clinical management of patients with ischaemic stroke.

Fungi Fibrinolytic Compound 1 Plays a Core Role in Modulating Fibrinolysis, Altering Plasma Clot Structure, and Promoting Susceptibility to Lysis.

Fibrin clot structure and function are major determinants of venous and arterial thromboembolic diseases, as well as the key determinants of the efficiency of clot lysis. Studies have revealed that fungi fibrinolytic compound 1 (FGFC1) is a novel marine pyranisoindolone natural product with fibrinolytic activity. Here, we explore the impacts of FGFC1 on clot structure, lysis, and plasminogen activation in vitro using turbidimetric, enzyme-linked immunosorbent assay, confocal and electron microscopy, urokinase, or plasmin chromogenic substrate. Clots formed in the presence of FGFC1 expressed reduced fibrin polymerization rate and maximum turbidity; however, they did not influence the lag phase of fibrin polymerization. In the absence of scu-PA (single-chain urokinase plasminogen activator), microscopy revealed that FGFC1 increased the number of protofibrils within fibrin fiber and the pore diameter between protofibrils, inducing clots to form a region of thinner and looser networks separated by large pores. The effects of FGFC1 on scu-PA-mediated plasma clot structure were similar to those in the absence of scu-PA. In addition, FGFC1 promoted the lysis of clots and increased the D-dimer concentration in lysate. FGFC1 increased the generation rate of p-nitroaniline in plasma. These results show that FGFC1 has fibrinolytic activity in plasma, leading to interference with the release of fibrinopeptide B to affect lateral aggregation of protofibrils and increase clot susceptibility to fibrinolysis by altering its structure.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

Cryoprecipitate transfusion in trauma patients attenuates hyperfibrinolysis and restores normal clot structure and stability: Results from a laboratory sub-study of the FEISTY trial.

BACKGROUND: Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. METHODS: Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. RESULTS: Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. CONCLUSIONS: In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

Why fibrin biomechanical properties matter for hemostasis and thrombosis.

Polymeric fibrin displays unique structural and biomechanical properties that contribute to its essential role of generating blood clots that stem bleeds. The aim of this review is to discuss how the fibrin clot is formed, how protofibrils make up individual fibrin fibers, what the relationship is between the molecular structure and fibrin biomechanical properties, and how fibrin biomechanical properties relate to the risk of thromboembolic disease. Fibrin polymerization is driven by different types of bonds, including knob-hole interactions displaying catch-slip characteristics, and covalent crosslinking of fibrin polypeptides by activated factor XIII. Key biophysical properties of fibrin polymer are its visco-elasticity, extensibility and resistance to rupture. The internal packing of protofibrils within fibers changes fibrin biomechanical behavior. There are several methods to analyze fibrin biomechanical properties at different scales, including AFM force spectroscopy, magnetic or optical tweezers and rheometry, amongst others. Clinically, fibrin biomechanical characteristics are key for the prevention of thromboembolic disorders such as pulmonary embolism. Future studies are needed to address unanswered questions regarding internal molecular structure of the fibrin polymer, the structural and molecular basis of its remarkable mechanical properties and the relationship of fibrin biomechanical characteristics with thromboembolism in patients with deep vein thrombosis and ischemic stroke.

Fibrin clot properties and fibrinolysis in patients with endocrine hypertension due to aldosterone or catecholamines excess.

OBJECTIVE: The aim of the study was to investigate a new possible background of increased risk of cardiovascular events in two forms of endocrine hypertension: in primary aldosteronism (PA) and pheochromocytoma/paraganglioma (PPGL) in comparison to essential hypertension (EHT). CONTEXT: Prothrombotic properties of the fibrin clot structure, impaired fibrinolysis and enhanced thrombin generation have been reported to be associated with increased cardiovascular risk. DESIGN: Patients with PA and PPGL were evaluated at baseline and re-evaluated 3 months after causative treatment. At baseline PA and PPGL patients were compared to matched EHT patients and to healthy controls. PATIENTS: The study included 35 patients with PA, 16 patients with PPGL and two reference groups of patients with EHT (32 and 22 patients) and healthy controls (35 and 23 subjects). MEASUREMENTS: All subjects underwent evaluation according to the study protocol that included plasma fibrin clot permeability (Ks), clot lysis time, endogenous thrombin potential. RESULTS: There were no differences in clot structure and fibrinolytic activity in PA and PPGL patients as compared to matched patients with EHT, whereas all hypertensive groups were characterized by more compact fibrin clot structure, faster clot formation and enhanced thrombin generation in comparison to healthy controls. Both in PA and PPGL patients, fibrin clot properties and fibrinolytic parameters remained stable after the causative treatment. CONCLUSIONS: Patients with PA and PPGL are at a prothrombic state comparable to patients with EHT. The results suggest the higher risk of cardiovascular events observed in hypertensive PA and PPGL as compared to EHT is not mediated through investigated prothrombic mechanisms.

The treatment effect of rivaroxaban on clot characteristics in patients who present acutely with first time deep vein thrombosis.

BACKGROUND: The acute vascular disease deep vein thrombosis (DVT) requires oral anticoagulants to prevent progression. Monitoring therapeutic efficacy of direct oral anticoagulants (DOAC), including rivaroxaban, is problematic as no reliable test is available. Advances in rheometry have led to the development of a functional coagulation biomarker using Gel Point (GP) analysis which assesses clot structure formation. The biomarker measures incipient clot formation time (TGP) and quantifies fibrin clot structure in terms of fractal dimension (df). OBJECTIVE: This study aimed to investigate clot structure formation in first time DVT and the effect of rivaroxaban treatment. METHODS: This prospective observational cohort study measured the GP and standard laboratory markers at three sample points: pre-treatment and at 20 and 60 days following 15 mg BD and 20 mg OD rivaroxaban respectively. RESULTS: Forty DVT patients (mean age 64 years [SD±14.8]; 23 males, 17 female) were recruited. The results show that DVT vs non-DVT patients did not have a significantly different GP profile (df: 1.72±0.06 vs 1.70±0.06 and TGP: 267±68 sec vs 262±73 sec) with both within the defined healthy index. In addition, rivaroxaban therapy increased TGP to 392 s (±135 s) after 20 days, and subsequently increased to 395 s (±194 s) at 60 days but did not significantly increase df (from 1.69±0.05 to 1.71±0.06). CONCLUSIONS: The results indicate in this cohort of DVT patients there was no underlying hypercoagulable effect as determined by gel point analysis. Furthermore, the anticoagulant effect of rivaroxaban prolonged clotting, suggesting a protective effect against clot formation, without significantly reducing clot microstructural properties.

Biomaterial Wettability Affects Fibrin Clot Structure and Fibrinolysis.

Thrombosis on blood-contacting medical devices can cause patient fatalities through device failure and unstable thrombi causing embolism. The effect of material wettability on fibrin network formation, structure, and stability is poorly understood. Under static conditions, fibrin fiber network volume and density increase in clots formed on hydrophilic compared to hydrophobic polystyrene surfaces. This correlates with reduced plasma clotting time and increased factor XIIa (FXIIa) activity. These structural differences are consistent up to 50 µm away from the material surface and are FXIIa dependent. Fibrin forms fibers immediately at the material interface on hydrophilic surfaces but are incompletely formed in the first 5 µm above hydrophobic surfaces. Additionally, fibrin clots on hydrophobic surfaces have increased susceptibility to fibrinolysis compared to clots formed on hydrophilic surfaces. Under low-flow conditions, clots are still denser on hydrophilic surfaces, but only 5 µm above the surface, showing the combined effect of the surface wettability and coagulation factor dilution with low flow. Overall, wettability affects fibrin fiber formation at material interfaces, which leads to differences in bulk fibrin clot density and susceptibility to fibrinolysis. These findings have implications for thrombus formed in stagnant or low-flow regions of medical devices and the design of nonthrombogenic materials.

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Patients scheduled for TAVI tend to form abnormal fibrin clots more resistant to lysis: the impact of age.

BACKGROUND: Fibrin accumulation within the stenotic leaflets associated with impaired fibrinolysis was observed in severe aortic stenosis (AS). Little is known about fibrin clot properties in patients scheduled for transcatheter aortic valve implantation (TAVI). AIMS: We investigated whether TAVI patients display a more prothrombotic state, including suppressed fibrinolytic capacity compared to those undergoing surgery. METHODS: We enrolled patients with advanced AS without significant atherosclerotic vascular disease scheduled for TAVI (n = 45) or surgical aortic valve replacement (SAVR, n = 59). Plasma fibrin clot features, including clot permeability (Ks) reflecting an average pore size, and lysis potential (Lys50), along with thrombin generation were determined off anticoagulation within 12 hours before the procedure. RESULTS: TAVI patients compared to SAVR had prolonged Lys50 (median 420 [IQR, interquartile range, 337-480] seconds vs 379 [337-428] seconds; P = 0.045) and formed denser clots, reflected by lower Ks (3.66 [3.05-4.84] vs 4.36 [3.6-5.27] × 10-9 cm2; P = 0.02), but after adjustment for age the latter difference was no longer significant. Apart from age, concomitant diabetes mellitus, or chronic kidney disease, prolonged Lys50 was an independent predictor of indication for TAVI in AS patients on multivariate regression analysis. There was a delayed start of thrombin generation in TAVI patients (lag time, 4.5 [3.8-6.3] minutes vs 4.2 [3.3-4.7] minutes; P = 0.035), without other differences in thrombin generation parameters. CONCLUSIONS: This study is the first to show that patients scheduled for TAVI are characterized by prothrombotic fibrin clot properties including denser fibrin meshwork and more resistant to lysis compared with those undergoing SAVR, which might explain in part increased thromboembolic risk following TAVI.

Effects of antithrombotic drugs on the prothrombotic state in patients with atrial fibrillation: The west Birmingham atrial fibrillation project.

BACKGROUND: Direct oral anticoagulants (DOACs) are known to prevent thrombosis but there is limited information about their activity on the clot formation and lysis cascade. OBJECTIVES: This study assesses the role of apixaban, one of the four licenced DOACs, on clot dynamics in patients with atrial fibrillation (AF). METHODS: We compared haemostatic and clot lysis characteristics between a group of patients with AF (n = 47) and a "disease control" group with ischaemic heart disease but in sinus rhythm (n = 39). Subsequently, we conducted clot structure studies in 3 groups of patients with AF on different antithrombotic drugs: warfarin (n = 60), apixaban (n = 60) or antiplatelets (n = 62) and in patients with AF naïve to oral anticoagulants before and after 3-months treatment with apixaban (n = 32). Haemostasis was investigated by a viscoelastic, whole blood technique (Thromboelastography/TEG), a "microplate-reader based", citrated plasma technique (microplate assay), immunoassays to determine plasma concentrations of plasminogen activator inhibitor-1 (PAI-1), tissue-Plasminogen Activator (t-PA), D-dimer and finally platelet derived and apoptotic microparticles. RESULTS: Patients with AF have more potent thrombogenesis based on microplate assay indices [Rate of clot formation (p = 0.03, ƞ2 = 0.06), Maximum optical density (p < 0.001, ƞ2 = 0.05)] and delayed fibrinolysis [Rate of clot dissolution (p = 0.005, ƞ2 = 0.17)] with increased levels of apoptotic microparticles (p = 0.02, ƞ2 = 0.06) compared with the 'disease control' group. Apixaban was more effective in attenuating prothrombotic characteristics assessed by TEG {R (ε2 = 0.21), K (ε2 = 0.16) and angle [mean difference (MD), 95% Confidence Intervals (CI), vs warfarin 5, 0.96-8.6 and 8, 3.8-11.4 vs antiplatelets], (p < 0.001 for all indices)} compared with the other treatment groups. Patients on apixaban had lower D-dimer (p < 0.001, ε2 = 0.17) and tPA (p = 0.03, MD 90, 95%CI 6-150 vs warfarin and MD 90, 95% CI 4-150 vs antiplatelets) levels. From the microplate assay analysis, warfarin and apixaban demonstrated comparable activity based on multiple indices, both superior to antiplatelets. However, warfarin was associated with reduced fibrin network robustness (Max. optical density p < 0.001, ε2 = 0.1). Apixaban inhibited thrombosis, amplified fibrinolysis and decreased D-dimer (p = 0.001, r = 0.4) levels in the follow up study. CONCLUSIONS: Patients with AF have impaired haemostasis and elevated levels of apoptotic microparticles. Apixaban appears to affect plasma prothrombotic characteristics in a distinctive manner compared with warfarin and to reduce biomarkers associated with adverse cardiovascular events.

Fibrin clot properties to assess the bleeding phenotype in unrelated patients with hypodysfibrinogenemia due to novel fibrinogen mutations.

Congenital hypodysfibrinogenemia is a rare fibrinogen disorder, defined by decreased levels of a dysfunctional fibrinogen. We present the functional and structural characterization of two new fibrinogen variants. A duplication of 32 bases in FGA exon 5, p.Ser382GlyfsTer50 was identified in a patient (P1) with history of hemoptysis and traumatic cerebral bleeding. A missense mutation in FGG exon 8, p.Ala353Ser was identified in two siblings (P2 and P3) with tendency to bruising and menorrhagia. Fibrin polymerization was studied in plasma and in purified fibrinogen by turbidimetry. Fibrin structure was studied by a permeability assay, laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). In both plasma and purified fibrinogen samples, all patients had an abnormal polymerization characterized by a decreased maximal absorption compared to controls. The permeation constant (Ks) was markedly increased in all patients: 31 ± 9 × 10-9 cm2 in P1, and 20 ± 0.1 × 10-9 cm2 in P2 and P3, compared to 6 ± 2 × 10-9 cm2 in the control (p < 0.05). The presence of very large pores that accounts for the increased Ks was confirmed by LSCM and SEM patients' clots images. By SEM, the patients' fibrin fibers diameters were thicker: 90 ± 25 nm in P1, 162 ± 64 nm in P2 and 132 ± 46 nm in P3 compared to 74 ± 25 nm in control (p < 0.0001). In conclusion, both new causative fibrinogen mutations altered clot structure by forming thick fibers, diminishing fiber branching, and increasing pore filling space. These structural changes to clots explain the patients' bleeding phenotypes.

Maturity onset diabetes of the young and fibrin-related thrombosis risk.

BACKGROUND: Fibrin network characteristics determine predisposition to cardiovascular disease (CVD). Individuals with type 1 (T1DM) and type 2 diabetes mellitus (T2DM) have higher risk of CVD and display deranged fibrin network structure. Those with maturity onset diabetes of the young (MODY) may also be at increased risk but their fibrin clot properties have not been studied. METHODS: Plasma clots properties from 13 individuals with HNF1A-MODY, 12 matched-individuals with T2DM and 12 with T1DM were studied using a validated turbidimetric assay and confocal microscopy. Plasma levels of fibrinogen, plasminogen activator inhibitor-1, complement C3 and C-reactive protein were also measured. RESULTS: MODY clot maximum absorbance was 0.37 ± 0.03 AU, similar to T1DM (0.32 ± 0.03 AU; p = 0.26), but lower than T2DM (0.49 ± 0.03 AU; p = 0.02), with confocal microscopy confirming structural differences. Clot lysis time in MODY was similar to T1DM (456 ± 50 and 402 ± 20 s, respectively; p = 0.09) but shorter than T2DM (588 ± 58 s; p = 0.006). Comparing inflammatory/thrombotic proteins in HNF1A-MODY and T2DM, C3 levels were lower in MODY than T2DM (0.58 ± 0.09 and 0.80 ± 0.1 mg/ml, respectively; p < 0.01). CONCLUSIONS: HNF1A-MODY fibrin network alterations are at least as pronounced as in T1DM but less thrombotic than T2DM clots. Differences in fibrin clot characteristics comparing HNF1A-MODY and T2DM may, in part, relate to lower C3 levels.

The Developing Balance of Thrombosis and Hemorrhage in Pediatric Surgery: Clinical Implications of Age-Related Changes in Hemostasis.

Bleeding and thrombosis in critically ill infants and children is a vexing clinical problem. Despite the relatively low incidence of bleeding and thrombosis in the overall pediatric population relative to adults, these critically ill children face unique challenges to hemostasis due to extreme physiologic derangements, exposure of blood to foreign surfaces and membranes, and major vascular endothelial injury or disruption. Caring for pediatric patients on extracorporeal support, recovering from solid organ transplant or invasive surgery, and after major trauma is often complicated by major bleeding or clotting events. As our ability to care for the youngest and sickest of these children increases, the gaps in our understanding of the clinical implications of developmental hemostasis have become increasingly important. We review the current understanding of the development and function of the hemostatic system, including the complex and overlapping interactions of coagulation proteins, platelets, fibrinolysis, and immune mediators from the neonatal period through early childhood and to young adulthood. We then examine scenarios in which our ability to effectively measure and treat coagulation derangements in pediatric patients is limited. In these clinical situations, adult therapies are often extrapolated for use in children without taking age-related differences in pediatric hemostasis into account, leaving clinicians confused and impacting patient outcomes. We discuss the limitations of current coagulation testing in pediatric patients before turning to emerging ideas in the measurement and management of pediatric bleeding and thrombosis. Finally, we highlight opportunities for future research which take into account this developing balance of bleeding and thrombosis in our youngest patients.

Emicizumab improves the stability and structure of fibrin clot derived from factor VIII-deficient plasma, similar to the addition of factor VIII.

INTRODUCTION: Emicizumab is an antifactor (F)IXa/FX bispecific antibody, mimicking FVIIIa cofactor function. Emi prophylaxis effectively reduces bleeding events in patients with haemophilia A. The physical properties of emicizumab-induced fibrin clots remain to be investigated, however. AIM: We have investigated the stability and structure of emicizumab-induced fibrin clots. METHODS: Coagulation was initiated by activated partial thromboplastin time (aPTT) trigger and prothrombin time (PT)/aPTT-mixed trigger in FVIII-deficient plasma with various concentrations of emicizumab or recombinant FVIII. The turbidity and stability of fibrin clots were assessed by clot waveform and clot-fibrinolysis waveform analyses, respectively. The resulting fibrin was analysed by scanning electron microscopy (SEM). RESULTS: Using an aPTT trigger, the turbidity was decreased and the fibrinolysis times were prolonged in the presence of emicizumab dose-dependently. Scanning electron microscopy imaging demonstrated that emicizumab improved the structure of fibrin network with thinner fibres than in its absence. Although emicizumab shortened the aPTT dramatically, the nature of emicizumab-induced fibrin clots did not reflect the hypercoagulable state. Similarly, using a PT/aPTT-mixed trigger that could evaluate potential emicizumab activity, emicizumab improved the stability and structure of fibrin clot in a series of experiments. In this circumstance, fibrin clot properties with emicizumab at 50 and 100 µg/mL appeared to be comparable to those with FVIII at ~12 and ~24-32 IU/dL, respectively. CONCLUSION: Emicizumab effectively improved fibrin clot stability and structure in FVIII-deficient plasma, and the physical properties of emicizumab-induced fibrin clots were similar to those with FVIII.

Fibrin γ/γ' influences the secretion of fibrinolytic components and clot structure.

BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

Glucose Concentration Affects Fibrin Clot Structure and Morphology as Evidenced by Fluorescence Imaging and Molecular Simulations.

Although in vivo studies have been conducted in the past to determine hyperglycemic effects and influence on clotting risk in patients with diabetes, the true extent of hyperglycemia on unstable and spontaneous clot formation remains highly debated. Factors such as increased glycation, elevated fibrinogen concentration, elevated prothrombin levels, and decreased plasminogen are known to influence fibrin conversion, clot morphology, and thrombus formation in these individuals. In this regard, the isolated effects of hyperglycemia on irregular fibrin clot formation were investigated in a controlled fibrinogen system. In this study, fibrin clot characteristic differences at 3 glucose concentrations were analyzed to determine the effects of glucose concentration on fibrinogen glycation and fibrin clot morphology using confocal microscopy, glycation quantification, molecular simulations, and image processing methods. Algorithms coupled with statistical analysis support in vivo findings that hyperglycemia increases fibrinogen glycation, with ensuing altered fibrin clot structure characteristics. Our experimental and molecular simulation results consistently show an increased glucose adsorption by fibrinogen with increased glucose concentration. Significant differences in clot structure characteristics were observed, and the results of this work can be used to further develop diagnostic tools for evaluating clotting risk in individuals with hypercoagulable and hyperglycemic conditions.