Dense Fibrin Network Research Articles

Increased levels of histidine-rich glycoprotein are associated with the development of post-thrombotic syndrome

Denser fibrin networks which are relatively resistant to lysis can predispose to post-thrombotic syndrome (PTS). Histidine-rich glycoprotein (HRG), a blood protein displaying antifibrinolytic properties, is present in fibrin clots. We investigated whether HRG may affect the risk of PTS in relation to alterations to fibrin characteristics. In venous thromboembolism (VTE) patients, we evaluated plasma HRG levels, plasma clot permeability, maximum absorbance, clot lysis time and maximum rate of increase in D-dimer levels released from clots after 3 months of the index event. We excluded patients with cancer and severe comorbidities. After 2 years of follow-up, 48 patients who developed PTS had 18.6% higher HRG at baseline. Baseline HRG positively correlated with clot lysis time, maximum absorbance, and thrombin-activatable fibrinolysis inhibitor (TAFI) activity but was inversely correlated with plasma clot permeability and maximum rate of increase in D-dimer levels released from clots. On multivariate regression model adjusted for age, fibrinogen and glucose, independent predictors of PTS were recurrent VTE, baseline HRG level, and TAFI activity. VTE recurred in 45 patients, including 30 patients with PTS, and this event showed no association with elevated HRG. Our findings suggest that increased HRG levels might contribute to the development of PTS, in part through prothrombotic fibrin clot properties.

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

AbstractAnticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre− mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

INFLUENCE OF RBC COUNT AND HAEMOGLOBIN CONCENTRATION ON PLATELET RICH FIBRIN NETWORK AMOMG DIFFERENT AGE GROUPS AND GENDER- A CROSS SECTIONAL STUDY.

AIM: To determine the influence of RBC count and hemoglobin concentration on the density of PRF network among individuals of different age groups and gender. MATERIALS AND METHODS: 82 individuals were divided into three groups based on their age, Group 1: 20-30 years, Group 2: 30-45 years, & Group 3: 45-60 years. 3-4 ml of venous blood samples were collected, PRF were prepared and it was subjected to histological analysis to determine the age and gender related changes in the density of PRF network. Hematological parameters such as Hb concentration and RBC count were also evaluated and correlated with the density of PRF network. RESULTS: Group 1 showed predominantly dense fibrin network, Group 2 showed mixed pattern including both dense and loose fibrin network and Group 3 showed predominantly loose fibrin network. However the difference in fibrin network among different age groups was not statistically significant (p value-0.14). Among the gender, males showed dense fibrin network compared to females which was statistically significant with p value-0.03. There was no statistically significant correlation between RBC count and density of fibrin network with p value-0.11. There was a statistically significant correlation between Hb level and density of fibrin network with p value-0.05. CONCLUSION: The PRF which is widely used in periodontal regenerative therapy differ in densities between male (dense network) and female (loose network). It is found to be denser in individuals with high hemoglobin concentration.

Silver-impregnated platelet-rich fibrin as a barrier membrane

Introduction: Platelet-rich fibrin (PRF) was developed to eliminate xenofactors form of platelet-rich plasma to be used as a source of growth factor for tissue regeneration. PRF has been used as an autologous grafting material because of its ability to accelerate physiologic wound healing and new bone formation. Aim: To compare mechanical and histologic characteristics of the PRF membrane and silver-impregnated PRF. Materials and Methods: Venous blood was taken from the subjects for PRF preparation. Then, 2 normal PRFs were prepared, and for silver-impregnated PRF, 9 ml of blood in addition to 1 ml of silver nanoparticle suspension was poured into another tube and gently shook with hand to achieve a uniform 1% concentration. The tubes were centrifuged at 3000 rpm for 10 min. The tensile test was done by the universal testing machine: The remaining pieces of the membranes were fixed in 10% formalin for 24 h to be subjected to hematoxylin and eosin staining and evaluated with the light microscope Degradation time. Results: Silver-impregnated showed improved mechanical properties and dense fibrin network than PRF. Conclusion: Silver-impregnated PRF membrane demonstrated properties to be used as a barrier membrane for periodontal reconstruction.

Dense and dangerous: The tissue plasminogen activator-resistant fibrinolysis shutdown phenotype is due to abnormal fibrin polymerization

Both hyperfibrinolysis and fibrinolysis shutdown can occur after severe trauma. The subgroup of trauma patients with fibrinolysis shutdown resistant to tissue plasminogen activator (t-PA)-mediated fibrinolysis have increased mortality. Fibrin polymerization and structure may influence fibrinolysis subgroups in trauma, but fibrin architecture has not been characterized in acutely injured subjects. We hypothesized that fibrin polymerization measured in situ will correlate with fibrinolysis subgroups. Blood samples were collected from trauma patients and noninjured controls. We selected samples across a range of fibrinolysis phenotypes (shutdown, physiologic, hyperfibrinolysis) and t-PA sensitivities (sensitive, physiologic, resistant) determined by thrombelastography. Plasma clots were created in situ with fluorescent fibrinogen and imaged using confocal microscopy for analysis of clot architecture in three dimensions. For each clot, we quantified the fiber resolvability, a metric of fiber distinctness or clarity, by mapping the variance of fluorescence intensity relative to background fluorescence. We also determined clot porosity by measuring the size and distribution of the gaps between fibrin fibers in three-dimensional space. We compared these measures across fibrinolysis subgroups. Fiber resolvability was significantly lower in all trauma subgroups compared with controls (n = 35 and 5, respectively; p < 0.05). We observed markedly different patterns of fibrin architecture among trauma patients stratified by fibrinolysis subgroup. Subjects with t-PA-resistant fibrinolysis shutdown exhibited abnormal, densely packed fibrin clots nearly devoid of pores. Individuals with t-PA-hypersensitive fibrinolysis shutdown had highly irregular clots with pores as large as 2500 μm to 20,000 μm, versus 78 μm to 1250 μm in noninjured controls. Fiber resolvability was significantly lower in trauma patients than controls, and subgroups of fibrinolysis differ in the porosity of the fibrin clot structure. The dense fibrin network in the t-PA-resistant group may prevent access to plasmin, suggesting a mechanism for thrombotic morbidity after injury.

Prothrombotic fibrin clot properties associated with increased endogenous thrombin potential and soluble P‐selectin predict occult cancer after unprovoked venous thromboembolism

BackgroundCompact fibrin clots relatively resistant to lysis are observed in patients at increased risk of venous thromboembolism (VTE) including malignancy. The citrullinated histone H3 (H3Cit) predicts VTE in cancer patients. ObjectivesWe performed a cohort study to investigate whether abnormal clot properties predict cancer diagnosis following unprovoked VTE. MethodsIn 369 consecutive patients aged <70 years without malignancy detected during routine screening, we determined plasma clot permeability (Ks) and clot lysis time (CLT), along with several prothrombotic markers and H3Cit after 2 to 8 months since VTE. ResultsDuring follow‐up (median, 37; interquartile range, 33‐39 months), malignancy was diagnosed in 22 patients (6%), who were older. This group had denser fibrin networks (−13% Ks), impaired fibrinolysis (+25.5% CLT), increased endogenous thrombin potential (ETP,+7%), soluble P‐selectin (+40.3%), and H3Cit (+169.2%) measured off anticoagulation after median 4 months since VTE. The Ks and CLT correlated with H3Cit (r= −.58 and r= .31, P< .05, respectively). The Kaplan–Meier survival analysis showed that reduced Ks (the first quartile, ≤6.2 × 10−9 cm2), prolonged CLT (the top quartile, >106 min), and higher ETP (the top quartile, >1657 nM × min) were predictors of cancer diagnosed during follow‐up. The multivariable Cox proportional hazards model showed that patients with the prothrombotic clot phenotype (low Ks and long CLT) had the highest risk of cancer diagnosis [hazard ratio(HR), 23.4; 95% confidence interval (CI), 6.67‐82.15]. ConclusionsProthrombotic clot properties following unprovoked VTE might help identify patients at risk of a diagnosis of cancer within the first 3 years of follow‐up.

Perfluorocarbon nanodroplets versus microbubbles in cavitation-enhanced sonothrombolysis of retracted clots

Microbubble-mediated sonothrombolysis has been effective in the treatment of acute clots (&amp;lt;24 h old), where microbubbles provide cavitation nuclei for cavitation-enhanced clot lysis. However, sonothrombolysis has shown very limited efficacy for the treatment of retreated clots (aged &amp;gt;3 days), as retracted clots have denser fibrin network which impedes the permeation of microbubbles. We hypothesize that the phase-change nanodroplets can permeate into retracted clots and improve sonothrombolysis. Phase-change nanodroplets are hundred-nanometer, liquid-filled contrast agents that convert to microbubbles upon excitation by thermal and acoustic energy. Due to their smaller size and higher stability than microbubbles, nanodroplets can provide longer circulation time and improved extravasation into less porous tissues. Here, we compared the thrombolytic effects of lipid shell-decafluorobutane microbubbles and similarly formulated nanodroplets in a flow model that contains a retracted bovine blood clot. Short burst ultrasound (1 MHz, 5.8 MPa, 0.5% duty cycle) was applied in three different therapy scenarios: ultrasound only, ultrasound with microbubbles, ultrasound with nanodroplets (n = 5). We observed internal clot erosion starting inside the clot samples by nanodroplet-mediated ultrasound, whereas the microbubble-mediated case resulted in only surface erosion. The nanodroplet-mediated treatment exhibited an averaged weight loss rate of 3.8 ± 0.1%/min, which was higher than the microbubble-mediated treatment (2.7 ± 0.15%/min).

Concentrated growth factor promotes proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells in vitro

ObjectiveThe aim of this research is to investigate the effects of concentrated growth factor (CGF) on the proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells (PDCs) in vitro.MethodsPDCs were isolated from the femoral and tibial periosteum of rabbits and cultured with or without CGF membranes or CGF conditioned media. Scanning electron microscopy (SEM) was used for the structural characterization. Cell Counting Kit-8 assay was used to measure cell proliferation. Alkaline phosphatase (ALP) activity of PDCs was also measured. Immunohistochemistry was used to detect the expression of CD34. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), and Western blot were used to evaluate the secretion and expression levels of osteogenic differentiation markers (bone morphogenetic protein-2, type I collagen, osteocalcin) and angiogenesis markers (vascular endothelial growth factor, basic fibroblast growth factor) in supernatants and PDCs at days 3, 7, 14, and 21.ResultsThe SEM analysis showed a dense three-dimensional fibrin network in CGF, and CGF membranes were covered by PDCs with elongated or polygonal morphological features. Compared with the control group, CGF significantly promoted the proliferation of PDCs during the experimental period (p < 0.05). Immunohistochemistry revealed that PDCs were dispersedly distributed among the CGF substrates, and CD34-positive cells were also present. Moreover, CGF significantly increased the ALP activity and upregulated the expression and secretion of osteogenic differentiation and angiogenesis markers in PDCs at days 3, 7, 14, and 21 (p < 0.05).ConclusionCGF can increase the proliferation and promote the osteogenic differentiation and angiogenic potential of PDCs in vitro. These results indicate that CGF can be used as a new therapeutic means for biotechnological and clinical applications.

The Hypofibrinolytic Defect of Nephrotic Syndrome Is Directly Proportional to Fibrin Network Density

IntroductionNephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Importantly, NS is associated with a complex acquired hypercoagulopathy and a high incidence (~25%) of life-threatening thrombotic complications.Both hypercoagulopathy and hypofibrinolysis are described contributors to NS-related VTE risk. However, the mechanisms underlying the latter are poorly understood. We previously showed NS disease severity is directly proportional to both hypercoagulopathy and fibrinolytic resistance There is evidence that fibrin clot structural density contributes to clot stability and has been observed in the presence of both increased plasma thrombin generation and fibrinogen levels, both of which we have previously demonstrated in NS.Thus the aim of the present study was to investigate the mechanistic relationship between fibrin clot structure and fibrinolysis using two rodent models of NS and a cohort of human NS patients. We hypothesized that hypofibrinolysis arises from increased fibrin network density in a manner directly proportional to NS disease severity.MethodsUsing two well-established rat models of NS, transgenic diphtheria toxin receptor (DTR) and puromycin aminonucleoside (PAN), we compared fibrinolytic markers to disease severity. A range of severity was induced by a single injection of diphtheria toxin (0-75 ng/kg IP) or PAN (0-150 mg/kg IV). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio (uPr:Cr). Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor and spun down to platelet poor plasma (PPP). Samples were also collected from a local cohort of pediatric and adult NS patients (n=23), along with the corresponding clinical lab data for each patient.Plasma clot lysis assay (CLA) was performed using urokinase (50 IU) +/- plasminogen (2.4 uM), on clots initiated with high (20 nM) or low (5 nM) thrombin. Clot fibrin network structure was visualized/assessed by laser scanning confocal microscopy using fluorescently-labeled fibrinogen as a tracer. Fibrinolytic markers in plasma were measured by ELISA.ResultsHypofibrinolysis: Previous findings of a hypofibrinolytic defect was confirmed with the CLA, such that plasma clot lysis at 60 min was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively. Additionally, plasma clot lysis by CLA was decreased in NS patients with uPrCr ≥2 (n=16) vs. <2 mg/mg (n=7) (96.1 vs 55.2 %, respectively; P=0.041). Similar results were found when the assay was repeated using high or low thrombin concentrations or increased UK (200 IU), with and without the addition of physiologic amounts of plasminogen. When the assay was performed in the absence of UK (0 IU), lysis at 60 min was drastically reduced (~17%) with no difference between groups.Mechanisms of Hypofibrinolysis: Fibrin network density increased with disease severity such that it was positively correlated with proteinuria (P=0.022) and negatively correlated with hypoalbuminemia (P=0.01) in our DTR rat model, with similar results seen in our human samples (Figure). As expected, fibrin network density was negatively correlated with plasma clot lysis (P=0.04), while plasma fibrinogen concentration (P=0.017), and thrombin generation (P=0.047) were positively correlated with fibrin density. There was no correlation with plasma uPA, PAI-1, a2AP, tPA, TAFI, or plasminogen.ConclusionsThese data suggest that nephrotic plasma forms thrombi with a denser fibrin network that is resistant to fibrinolysis, in a manner that is proportional to disease severity. The significant correlation between thrombin generation and fibrin network density suggest that plasma thrombotic potential may be a key mechanism contributing to the altered clot structure and impaired clot lysis of NS. Current studies are exploring the mechanisms underlying and in vivo significance of fibrinolytic resistance in our rat NS models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antithrombotic medications and their impact on fibrin clot structure and function.

Fibrin constitutes a major protein component of intravascular thrombi in all locations. Fibrin formation and its functions are essential for physiological hemostasis and the pathologic thrombosis. Formation of dense fibrin networks which are relatively resistant to lysis is observed in patients with venous or arterial thromboembolism, including myocardial infarction, ischemic stroke and venous thromboembolism. Measures of clot characteristics, in particular clot permeability and clot lysis time, may predict arterial and venous recurrent thromboembolic events. Medications, including vitamin K antagonists (VKA), direct oral anticoagulants (DOAC), and parenteral direct or indirect thrombin or activated factor X inhibitors increase clot permeability, reflecting fibrin network density, in association with enhanced efficiency of fibrinolysis. These effects are only in part related to decreased thrombin generation. There is evidence that aspirin can also favorably alter fibrin clot properties probably through acetylation of fibrinogen. No such effects were observed for P2Y12 inhibitors. Of note, plasma fibrin clot permeability has been shown to predict adverse clinical outcomes in patients receiving oral anticoagulants, which might have practical implications. The current review summarizes data on effects of antithrombotic agents on fibrin clot phenotype in cardiovascular disease.

Altered Plasma Clot Properties and Trauma-Related Venous Thromboembolism despite Thromboprophylaxis.

Prothrombotic clot phenotype may characterize patients developing deep vein thrombosis (DVT) despite pharmacological thromboprophylaxis. We studied the role of fibrin clot properties and its potential determinants in individuals who experienced DVT after lower limb injury. In a case-control study, we assessed 50 patients who developed DVT despite prophylactic use of low-molecular-weight heparins (the failed thromboprophylaxis group) after a lower limb injury, and three age- and sex-matched control groups, 50 patients each: (1) patients with trauma-related DVT without prior thromboprophylaxis; (2) individuals with unprovoked DVT; (3) patients without history of DVT (the no-DVT controls). Fibrin clot properties, along with thrombin concentration and α2-antiplasmin, were assessed following 3 months of anticoagulation in all DVT patients. Compared with the no-DVT controls, the failed thromboprophylaxis group exhibited denser fibrin networks (12.8% lower clot permeability [Ks], p = 0.0008) and impaired fibrinolysis (46.2% longer clot lysis time [CLT], p = 0.0001 and 8% lower rate of D-dimer release from clots, p = 0.0008). In the unprovoked DVT, similar Ks and 14.9% shorter CLT (p = 0.02) were reported compared with the failed thromboprophylaxis group. The failed thromboprophylaxis patients had higher odds of having elevated peak thrombin generation (>241.5 nM, 90th percentile in the no-DVT controls; odds ratio [OR]: 3.62; 95% confidence interval [CI], 1.86-7.06; p = 0.002), and higher odds of having elevated α2-antiplasmin (>115.05%; OR: 3.38; 95% CI, 1.64-6.98; p = 0.001). Patients who experienced DVT despite thromboprophylaxis following lower limb trauma display a strongly prothrombotic fibrin clot phenotype, including increased clot density and hypofibrinolysis associated with higher plasma α2-antiplasmin.

Biomimetic Implant Surface Functionalization with Liquid L-PRF Products: In Vitro Study.

Objective Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. Methods Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). Results Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. Conclusions Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products.

Controlling Fibrin Network Morphology, Polymerization, and Degradation Dynamics in Fibrin Gels for Promoting Tissue Repair.

Fibrin is an integral part of the clotting cascade and is formed by polymerization of the soluble plasma protein fibrinogen. Following stimulation of the coagulation cascade, thrombin activates fibrinogen, which binds to adjacent fibrin(ogen) molecules resulting in the formation of an insoluble fibrin matrix. This fibrin network is the primary protein component in clots and subsequently provides a scaffold for infiltrating cells during tissue repair. Due to its role in hemostasis and tissue repair, fibrin has been used extensively as a tissue sealant. Clinically used fibrin tissue sealants require supraphysiological concentrations of fibrinogen and thrombin to achieve fast polymerization kinetics, which results in extremely dense fibrin networks that are inhibitory to cell infiltration. Therefore, there is much interest in developing fibrin-modifying strategies to achieve rapid polymerization dynamics while maintaining a network structure that promotes cell infiltration. The properties of fibrin-based materials can be finely controlled through techniques that modulate fibrin polymerization dynamics or through the inclusion of fibrin-modifying biomaterials. Here, we describe methods for characterizing fibrin network morphology, polymerization, and degradation (fibrinolysis) dynamics in fibrin constructs for achieving fast polymerization dynamics while promoting cell infiltration.

Altered plasma fibrin clot properties in hypertensive patients with obstructive sleep apnoea are improved by continuous positive airway pressure treatment.

We investigated plasma fibrin clot properties in high-risk hypertensive patients with obstructive sleep apnoea (OSA) and assessed the impact of continuous positive airway pressure (CPAP) treatment on clot phenotype. We studied 50 hypertensive patients with clinically significant OSA (age 50.0 ± 8.8 years, 39 M, 11 F). In total, 38 hypertensive patients without OSA balanced for age, sex, blood pressure, cardiovascular risk factors, and metabolic status served as controls. Plasma fibrin clot properties, including clot permeability coefficient, clot lysis time (CLT), and turbidimetric parameters of clot formation were determined. Patients underwent transthoracic echocardiography, carotid ultrasonography, evaluation of endothelial function and calcium score index of coronary arteries, and Doppler imaging of renal arteries. Compared with controls, OSA patients were characterized by more compact fibrin structure (lower median clot permeability coefficient, 6.00 vs. 7.25 10 cm; P < 0.001), impaired fibrinolysis (longer median CLT, 108.00 vs. 92.50 min; P < 0.001), and by faster clot formation (shorter median lag phase, 40.50 vs. 42.50 s; P = 0.041), and higher median maximum clot absorbency indicating denser fibrin networks (0.87 vs. 0.81; P = 0.028). Clot permeability coefficient and CLT correlated with apnoea-hypopnoea index (r = -0.46; P < 0.001 and r = 0.44; P < 0.001, respectively) as well with mean (r = 0.31; P = 0.003; r = -0.36; P = 0.001, respectively) and minimal oxygen saturation (r = 0.46; P < 0.001; r = -0.49; P < 0.001, respectively). After 3 months of CPAP treatment we observed an increase in clot permeability coefficient (5.95 vs. 7.60 10 cm; P = 0,001), shortened CLT (107.00 vs. 87.00; P = 0.006), a longer lag phase of fibrin formation (40.00 vs. 43.50 s; P = 0.013), and a trend toward lower maximum clot absorbency (0.86 vs. 0.81; P = 0.058). In hypertensive patients at high cardiovascular risk, OSA was associated with unfavourable prothrombotic fibrin clot characteristics, including hypofibrinolysis, which significantly improve as early as after 3 months of CPAP treatment.

Root Conditioning with Citric Acid and Ethylenediaminetetraacetic Acid and their Effect on Fibrin Clot Adhesion to Dentin-A Scanning Electron Microscopic Study.

Root biomodifiers are used to remove the smear layer formed on the roots as a result of mechanical debridement and also to expose collagen fibers making the root surfaces biologically acceptable. Further, the blood element linkage to demineralized roots and clot stabilization by collagen fibres are vital for the wound healing and success of periodontal surgery. The aim of this study was to evaluate and compare the distribution of fibrin network to dentin surfaces conditioned with citric acid and Ethylenediaminetetraacetic Acid (EDTA) using Scanning Electron Microscope (SEM). Thirty human dentin blocks (4 mm x 3 mm x 1 mm) were prepared and equally divided into three groups. The control Group A specimens were treated with Phosphate Buffered Saline (PBS). The study Group B and C specimens were treated with citric Acid and EDTA respectively. After conditioning, a drop of fresh human whole blood was placed on the dentin specimens and was allowed to clot. The specimens were then processed using standardized protocol for SEM. The dentin surfaces were then analysed for fibrin network linkage under SEM and scored according to the degree of fibrin linkage to the dentin surface. Statistical analysis was done using Kruskal Wallis test to compare the score values between the study groups. Pairwise comparison between the study groups was done using Mann Whitney U test with Bonferroni correction. The fibrin network formed on the dentin network were analysed and scores were given. Kruskal Wallis test and Mann Whitney U test with Bonferroni correction were used for statistical analysis. It was found that most of the dentin specimens conditioned with citric acid and covered with human blood exhibited dense fibrin network compared to EDTA. However, statistical analysis showed that there were no significant differences found between the groups (p=0.35). Citric acid and EDTA has almost comparable effect in promoting the adhesion of fibrin network onto the dentin surface which may further enhance connective tissue attachment to the root surface.

Blocking Protein S Improves Hemostasis in Hemophilia a and B

Introduction&AimHemophilia A and B are X-linked disorders caused by an absence or a reduction in coagulation FVIII and respectively, FIX. Patients with hemophilia often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis.Here, we investigated whether targeting protein S (PS) could promote hemostasis in hemophilia by re-balancing coagulation. PS is a natural anticoagulant, acting as non-enzymatic cofactor for activated protein C (APC) in the inactivation of FVa and FVIIIa, and for tissue factor pathway inhibitor (TFPI) in the inhibition of FXa. This dual role makes PS a key regulator of the inhibition of thrombin generation (TG).Methods & ResultsHemophilic (F8-/- or F9-/-) Pros1+/- mice were intercrossed. F8-/-Pros1-/- and F9-/- Pros1-/- mice were viable and found at the expected Mendelian frequency with no increased mortality compared to hemophilic mice.F8-/-Pros1-/- mice did not show DIC onset neither an increased mortality once challenge with tissue factor (TF). Ex vivo evaluation of TG potential showed that F8-/-Pros1-/- mice were APC-resistant, they had an improved low TF induced-TG and a 20% more dense fibrin network with larger fibrin fibers diameter as compared to F8-/-mice. Comparable results were found in human HA plasma where blocking PS raised TG even in the presence of high inhibitor titer.To assess the in vivo effect of PS inhibition on HA mice hemostasis, two tail- clipping (TC) assays were used. In both, mild TC model (2 mm cut) and severe TC (4 mm cut), blood loss significantly decreased in F8-/-Pros1-/- compared to F8- /- mice (mild TC: 407±21 vs 661±29ul, p<0.0001; severe TC:173±14 vs 249±24ul, p<0.05). In addition, the infusion of anti-hPS antibody on F8-/-Pros1+/- mice, reduced the blood loss compared to F8-/-Pros1+/- mice infused with an isotype IgG (196±10 vs 302±25ul, p=0.005).As recurrent joint bleeding is the most common manifestation of HA, we challenged F8-/-Pros1-/- mice in an acute hemarthrosis (AH) model. Joint swelling 72 hours after injury was reduced in F8-/-Pros1-/- compared to F8-/- (0.11±0.03 vs 1.02±0.07mm, p<0.0001, n=10). These results were also confirmed by s.c. injection of anti-hPS antibody (0.46±0.0 vs 0.78±0.09mm isotype IgG, p=0.02, n=9) and by i.v. injection of PS-siRNA prior to AH challenge in F8-/-Pros1+/- (0.29±0.09 vs 0.92±0.12mm siRNA control, p=0.03, n=5) and F8-/- mice(0.35±0.08 vs 0.78±0.09mm siRNA control, p=0.05, n=5). Similar results were obtained in F9-/-Pros1-/- mice. Histological analysis of joint showed joint bleeding reduction in F8-/-Pros1-/- compared to F8-/- and an increased fibrin staining comparable to F8+/+ mice.To understand the intra-articular hemostatic effect of blocking PS, joint sections were stained for TFPI. Preliminary results indicate a massive staining in the synovia of F8-/- mice, while F8-/-Pros1-/- and F8+/+ mice present a less intense signal. These data suggest that the intra-articular space is a modulable anticoagulant environment. Human HA joint tissues were then analyzed for both PS and TFPI. A strong signal was found for TFPI and PS in the synovial lining and sublining layers of HA patients on demand (n=7). Interestingly, PS and TFPI stainings were remarkably decreased in HA patients under prophylaxis (n=5). Joint section from osteoarthritis patients (n=7) did not show an intense staining for TFPI and PS similarly to hemophilic patients under prophylaxis.To understand the improved phenotype of F8-/-Pros1-/- after AH, the function of macrophages were investigated. At the steady state, F8-/-Pros1-/- presented significantly 2-fold increased levels of inflammatory macrophages (M1) than in F8-/- mice. In addition bone marrow derived macrophages from F8-/-Pros1-/- exhibit 10-fold higher RBC phagocytic activity than F8-/- . Preliminary results indicate an increase of a monocyte attractant MCP-1 level, in knee lavage after AH in F8-/-Pros1-/- than F8-/- mice.ConclusionThese data provide the first evidence that blocking PS has the ability to ameliorate hemophilia as judged by in vivo improvement of bleeding phenotype in the TC assay as well as in the AH model, suggesting a new valuable tool for hemophilia therapy. In addition, the modulable presence of PS and TFPI in human and mice joints is a novel pathophysiological aspect of hemarthrosis and constitutes a potential therapeutical target. DisclosuresKremer Hovinga:NovoNordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL-Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Leukocyte XII Regulates Venous Thrombosis Risk

Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis.Methods & Results: Sterile punch biopsy wounds were created on wild type (WT) and F12-/- mice. On Days 2 and 5, there was a ~3-fold decrease in CD11b-stained cells in F12-/- woundsvs. WT. On the thioglycolate (TG)-induced sterile peritonitis assay, lavage fluid from F12-/- mice contained significantly less peritoneal exudative cells (PEC)] on days 1 and 7, (p<0.008). To determine the contribution of XII in WBC function, we used XII siRNA (Alnylam Pharmaceuticals) to create plasma XII deficiency in WT mice. After tail vein injection, plasma XII was reduced to < 5% within 24 h (T1/2 plasma XII: 6.7 h). In the TG assay, even though plasma XII is decreased to less than 5%, PEC migration is the same as in WT mice. These data suggest that the reduced leukocyte migration observed in F12-/- mice is related to altered leukocyte function. On adoptive bone marrow (BM) transplantation (BMT) experiments, WT BM transplanted into KO hosts corrects the leukocyte migration defect on the TG assay. These data suggest that there is a pool of XII associated with BM cells that is functionally distinct than plasma-derived, hepatic XII. F12 cDNA is found in leukocytes and shares sequence homology to hepatic XII. Immunofluorescence confirms XII antigen on murine BM-derived and human peripheral blood WBCs. No XII antigen is observed in BM-derived leukocytes from F12-/- mice. When WBC are activated with fMLF, XII antigen translocates to the external membrane. F12-/- PMNs have reduced chemotaxis to fMLF and adherence to several integrin-binding glycoproteins. pAktS473 mediates neutrophil cell migration, integrin activation, and cytoskeletal assembly. Normal and F12-/- PMNs exhibit pAktS473 in response to fMLF and XII. Histologically, F12-/- wounds show a smaller wound gap and a greater percentage of wound re-epithelialization than WT controls. Inferior vena cava (IVC) thrombosis induced by 90% restriction to flow at 24h contains a smaller thrombus in F12-/- than WT mice (p<0.04). Histologically, IVC thrombi from WT mice contain abundant neutrophils that are adherent to the wall and trapped within a dense fibrin network (Fig 1). siRNA treatment results in less-occlusive thrombi (n.s) with an adequate neutrophil content but a finer fibrin network (Fig 1). F12-/- thrombi are non-occlusive and contain significantly less adherent neutrophils (Fig 1). XII itself is integrally a part of neutrophil extracellular traps (NETs) in the forming thrombus and F12-/- mice have reduced NETs at sites of occlusion. WT BM transplanted into F12-/- hosts corrects the thrombus weight and degree of inflammation in F12-/- mice to normal. Likewise, F12-/- BM into WT hosts, reduces thrombus weight and degree of inflammation.Conclusions: Leukocyte XII has a dual role in neutrophil function. We hypothesize that signaling by leukocyte XII contributes to neutrophil trafficking in sites of inflammation and venous stasis. At these sites, neutrophils become indispensable for activation of both the extrinsic and intrinsic pathways of coagulation during the early formation of intraluminal fibrin and for subsequent thrombus propagation by NETs and the activation of circulating XII. Defining the signaling pathway of XII in leukocytes will further our understanding as to the mechanism(s) by which these cells cooperate to initiate and propagate venous thrombosis in vivo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Contribution of Red Blood Cells and Clot Structure to Thrombosis

The blood clot or thrombus is composed of red blood cells, platelets and white cells that interact with the fibrin meshwork produced by the coagulation system. The red blood cells form polyhedrocytes that seal the clot, platelets provide forces for clot contraction, and white cells contribute neutrophil extracellular traps, cytokines and complement activation. The fibrin network contributes clot elasticity and provides the proteinaceous backbone structure that stabilises the clot. Previous studies from our laboratory and others have shown that dense fibrin networks demonstrating small pores and increased resistance to fibrinolysis associate with thrombosis. However, the mechanisms underpinning this have not been fully understood. Recent studies using atomic force microscopy from our laboratory have shown that fibrinogen interacts with red cells with comparable affinity as that with platelets. A patient with mutations in the β3 integrin subunit showed no binding between red cells and fibrinogen, demonstrating that a β3-related integrin receptor is involved in the interaction. Mutations in the fibrinogen α-chain integrin binding sites (D97E and D574E) reduced frequency of red cell interactions with fibrinogen. Interestingly, a naturally occuring splice variant of the fibrinogen γ-chain that reduces binding to the platelets, fibrinogen γ', increased binding interactions between fibrinogen and red blood cells. Fibrinogen γ' is a naturally occurring splice variant of fibrinogen, in which the C-terminal AGDV residues of the more common γA-chain (85%) are replaced with a negatively charched VRPEHPAETEYDSLYPEDDL sequence of the γ' chain (15%). Fibrinogen γ' induced clustering of fibrin fibres into tightly interknit nuclei of fibrin fibres, interspersed by large pores that extend over more than 50 μm within the fibrin network structure. The effects of fibrinogen γ' on fibrin clot structure was independent of thrombin and FXIII as demonstrated using snake venom enzyme. Previously we showed impaired fibrin protofibril formation with fibrinogen γ' using atomic force microscopy. Using turbidimetric analysis of fibrin intrafibrillar structure, we show that fibrinogen γ' reduces protofibil packing per fibrin fiber. Furthermore, we find that reduced protofibril packing diminishes fibrin stiffness as analysed with magnetic tweezers both in purified systems as well as in plasma at (patho)physiological fibrinogen γ' levels that range from 3-40%, and in whole blood as analysed with thromboelastography. In conclusion, our data show that red blood cells and fibrinogen γ' play major roles in the regulation of clot structure and stability, and that these effects on clot structure are major determinants of the functional properties of the blood clot. Modulating fibrin clot structure and its interactions with blood cells may represent major new targets for the treatment of thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Denser plasma clot formation and impaired fibrinolysis in paroxysmal and persistent atrial fibrillation while on sinus rhythm: Association with thrombin generation, endothelial injury and platelet activation

IntroductionFormation of compact and poorly lysable fibrin clots have been demonstrated in patients following ischemic stroke. Recently, it has been shown that denser fibrin networks and impaired fibrinolysis occurs in subjects with permanent atrial fibrillation (AF). Fibrin clot phenotype in other types of AF remains to be established. We evaluated fibrin clot properties in paroxysmal (PAF) and persistent AF (PsAF). Material and MethodsWe studied 88 non-anticoagulated patients with AF on sinus rhythm and free of stroke (41 with PAF, 47 with PsAF) versus 50 controls. Ex-vivo plasma fibrin clot permeability (Ks) and clot lysis time (CLT) were evaluated along with von Willebrand factor (vWF), peak thrombin generation (TG), platelet factor 4 (PF4) and fibrinolytic proteins. ResultsCompared with control subjects, clots obtained from plasma of patients with PAF and PsAF had similarly lower Ks (−7.7%, P=0.01; −8.6%, P=0.005, respectively) and prolonged CLT (+10.8%, P=0.006; +7.8% P=0.04, respectively). No associations of Ks and CLT with CHA2DS2-VASc and HAS-BLED score were observed. Patients with AF had higher TG, vWF, PF4 and plasminogen activator inhibitor-1 (PAI-1) antigen compared with controls. Multiple linear regression adjusted for age, gender, body mass index and fibrinogen showed that TG (β=−0.41), vWF (β=−0.29) and PF4 (β=−0.28) are the independent predictors of Ks (R2=0.78), while CLT was independently predicted by TG (β=0.37), PAI-1 antigen (β=0.29) and vWF (β=0.26) in the AF group (R2=0.39). ConclusionsPatients with PAF and PsAF while on sinus rhythm display unfavorably altered fibrin clot properties, which might contribute to thromboembolic complications.

Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood

Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-Hphob) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-HphilNS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-HphilNS but only in single patches of fibrin and small isolated fibre complexes on SLA-Hphob. On SLActive-HphilNS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-HphilNS compared to SLA-Hphob. The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.