ResearchPad - fibrin https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A three-dimensional phase-field model for multiscale modeling of thrombus biomechanics in blood vessels]]> https://www.researchpad.co/article/elastic_article_14644 Thromboembolism is associated with detachment of small thrombus pieces from the bulk in the blood vessel. These detached pieces, also known as emboli, travel through the blood flow and may block other vessels downstream, e.g., they may plug the deep veins of the leg, groin or arm, leading to venous thromboembolism (VTE). VTE is a significant cause of morbidity and mortality and it affects more than 900,000 people in the United States and result in approximately 100,000 deaths every year. Mechanical interaction between flowing blood and a thrombus is crucial in determining the deformation of the thrombus and the possibility of releasing emboli. In this study, we develop a phase-field model that is capable of describing the structural properties of a thrombus and its biomechanical properties under different blood flow conditions. Moreover, we combine this thrombus model with a particle-based model which simulates the initiation of the thrombus. This combined framework is the first computational study to simulate the development of a thrombus from platelet aggregation to its subsequent viscoelastic responses to various shear flows. Informed by clinical data, this framework can be used to predict the risk of diverse thromboembolic events under physiological and pathological conditions.

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<![CDATA[Comparisons of early vascular reactions in biodegradable and durable polymer-based drug-eluting stents in the porcine coronary artery]]> https://www.researchpad.co/article/5c40f823d5eed0c48438714e

Current drug-eluting stents have abluminal polymer coating; however, thrombus formation in these compared with that in uniformly coated stents remains controversial. We evaluated thrombus formation and early endothelialization after using abluminal biodegradable polymer-coated sirolimus- (BP-SES), and everolimus-eluting stents (BP-EES) versus a durable polymer-coated everolimus-eluting stent (DP-EES) in an in vivo setting. BP-SES, BP-EES, and DP-EES (n = 6 each) were implanted in coronary arteries of 12 mini-pigs that were then sacrificed after 7 and 10 days. Stents were stained with hematoxylin and eosin, and a combined Verhoeff and Masson trichrome stain. Areas of fibrin deposition were digitally detected and measured with off-line morphometric software. Stents were investigated for re-endothelialization by transmission electron microscopy. At 7 days, histological analysis revealed the lowest area of fibrin deposition in BP-SES (BP-SES vs. BP-EES vs. DP-EES; 0.10 ± 0.06 mm2 vs. 0.15 ± 0.07 mm2 vs. 0.19 ± 0.06 mm2, p = 0.0004). At 10 days, the area of fibrin deposition was significantly greater in DP-EES (0.13 ± 0.04 mm2 vs. 0.14 ± 0.05 mm2 vs. 0.19 ± 0.08 mm2, p = 0.007). Endothelial cells in BP-SES demonstrated a significantly greater number of tight junctions than those in DP-EES according to by transmission electron microscopy for both days (p<0.05). Various parameters, including an inflammatory reaction and neointimal formation, were comparable among the groups at 7 and 10 days. An abluminal biodegradable polymer-coated SES showed the least fibrin deposition and greatest endothelial cell recovery at an early stage following implantation in the coronary arteries of mini-pigs.

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<![CDATA[Effect of pathological heterogeneity on shear wave elasticity imaging in the staging of deep venous thrombosis]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be108f

Background

We aimed to observe the relationship between the pathological components of a deep venous thrombus (DVT), which was divided into three parts, and the findings on quantitative ultrasonic shear wave elastography (SWE) to increase the accuracy of thrombus staging in a rabbit model.

Methods

A flow stenosis-induced vein thrombosis model was used, and the thrombus was divided into three parts (head, body and tail), which were associated with corresponding observation points. Elasticity was quantified in vivo using SWE over a 2-week period. A quantitative pathologic image analysis (QPIA) was performed to obtain the relative percentages of the components of the main clots.

Results

DVT maturity occurred at 2 weeks, and the elasticity of the whole thrombus and the three parts (head, body and tail) showed an increasing trend, with the Young's modulus values varying from 2.36 ± 0.41 kPa to 13.24 ± 1.71 kPa; 2.01 ± 0.28 kPa to 13.29 ± 1.48 kPa; 3.27 ± 0.57 kPa to 15.91 ± 2.05 kPa; and 1.79 ± 0.36 kPa to 10.51 ± 1.61 kPa, respectively. Significant increases occurred on different days for the different parts: the head showed significant increases on days 4 and 6; the body showed significant increases on days 4 and 7; and the tail showed significant increases on days 3 and 6. The QPIA showed that the thrombus composition changed dynamically as the thrombus matured, with the fibrin and calcium salt deposition gradually increasing and the red blood cells (RBCs) and platelet trabecula gradually decreasing. Significant changes were observed on days 4 and 7, which may represent the transition points for acute, sub-acute and chronic thrombi. Significant heterogeneity was observed between and within the thrombi.

Conclusions

Variations in the thrombus components were generally consistent between the SWE and QPIA. Days 4 and 7 after thrombus induction may represent the transition points for acute, sub-acute and chronic thrombi in rabbit models. A dynamic examination of the same part of the thrombus may be helpful for improving the sensitivity and reproducibility of SWE for DVT diagnosis and staging.

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<![CDATA[An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbda7

The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.

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<![CDATA[Plasma fibrin D-dimer and the risk of left atrial thrombus: A systematic review and meta-analysis]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc570

Background

Plasma fibrin d-dimer has been taken as a marker for thrombus. The aim of this study was to evaluate the relationship between d-dimer (DD) levels and left atrial spontaneous echo contrast (SEC)/left atrial thrombus (LAT).

Methods

We identified clinical studies by systematic search of MEDLINE and EMBASE databases up to Feb 2016. All observational studies that considered DD as a study factor and trans-esophageal echocardiography (TEE) identified SEC/LAT as an outcome were included. Two reviewers independently selected the studies and extracted the data.

Results

Of the 21 included studies, 16 studies (2652 patients) have compared the mean DD differences between patients with and without an evidence of the presence of SEC/LAT, 9 studies (1667 patients) have estimated the diagnostic value of DD in the presence of LAT, and 11 studies (1856 patients) have available information to calculate a ratio of the presence of LAT among individuals in the top and the bottom third of DD levels. The pooled standardized mean difference (SMD) of DD between patients with and without left atrial SEC and/or LAT was 1.29 [95%CI: 0.51, 2.08], with SMDs of 0.42 [95% CI: 0.08, 0.77] and 2.34 [95% CI: 1.01, 3.68] in SEC/LAT and LAT subgroups, respectively. The combined risk ratio of the presence of LAT among individuals between the top of the distribution of DD levels and that in the bottom third was 3.84 [95% CI: 2.35, 6.28], associating with a mean difference of 0.78 ug/ml (1.10 vs 0.32 ug/ml). The pooled sensitivity, specificity and positive likelihood ratio of DD for LAT were 0.75 [95% CI: 0.65, 0.83], 0.81 [95% CI: 0.59, 0.93] and 4.0 [95% CI: 1.7, 9.9], respectively.

Conclusions

High plasma fibrin DD was associated with left atrial SEC/LAT, particularly among patients with LAT. DD levels have moderate sensitivity and specificity for diagnosing LAT.

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<![CDATA[Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications]]> https://www.researchpad.co/article/5989d9dcab0ee8fa60b67d6f

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

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<![CDATA[Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling]]> https://www.researchpad.co/article/5989db59ab0ee8fa60bdf0f0

Pericytes regulate key neurovascular functions of the brain. Studies in pericyte-deficient transgenic mice with aberrant signaling between endothelial-derived platelet-derived growth factor BB (PDGF-BB) and platelet-derived growth factor receptor β (PDGFRβ) in pericytes have contributed to better understanding of the role of pericytes in the brain. Here, we studied PdgfrβF7/F7 mice, which carry seven point mutations that disrupt PDGFRβ signaling causing loss of pericytes and vascular smooth muscle cells (VSMCs) in the developing brain. We asked whether these mice have a stable or progressive vascular phenotype after birth, and whether both pericyte and VSMCs populations are affected in the adult brain. We found an early and progressive region-dependent loss of brain pericytes, microvascular reductions and blood-brain barrier (BBB) breakdown, which were more pronounced in the cortex, hippocampus and striatum than in the thalamus, whereas VSMCs population remained unaffected at the time when pericyte loss was already established. For example, compared to age-matched controls, PdgfrβF7/F7 mice between 4–6 and 36–48 weeks of age developed a region-dependent loss in pericyte coverage (22–46, 24–44 and 4–31%) and cell numbers (36–49, 34–64 and 11–36%), reduction in capillary length (20–39, 13–46 and 1–30%), and an increase in extravascular fibrinogen-derived deposits (3.4–5.2, 2.8–4.1 and 0–3.6-fold) demonstrating BBB breakdown in the cortex, hippocampus and thalamus, respectively. Capillary reductions and BBB breakdown correlated with loss of pericyte coverage. Our data suggest that PdgfrβF7/F7 mice develop an aggressive and rapid vascular phenotype without appreciable early involvement of VSMCs, therefore providing a valuable model to study regional effects of pericyte loss on brain vascular and neuronal functions. This model could be a useful tool for future studies directed at understanding the role of pericytes in the pathogenesis of neurological disorders associated with pericyte loss such as vascular dementia, Alzheimer’s disease, amyotrophic lateral sclerosis, stroke and human immunodeficiency virus-associated neurocognitive disorder.

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<![CDATA[Vascular Endothelial Cell Injury Is an Important Factor in the Development of Encapsulating Peritoneal Sclerosis in Long-Term Peritoneal Dialysis Patients]]> https://www.researchpad.co/article/5989da0aab0ee8fa60b77567

Background and Objectives

Encapsulating peritoneal sclerosis (EPS) is a rare but serious and life-threatening complication of peritoneal dialysis (PD). However, the precise pathogenesis remains unclear; in addition, predictors and early diagnostic biomarkers for EPS have not yet to be established.

Methods

Eighty-three peritoneal membrane samples taken at catheter removal were examined to identify pathological characteristics of chronic peritoneal deterioration, which promotes EPS in patients undergoing long-term PD treatment with low occurrence of peritonitis.

Results

According to univariable logistic regression analysis of the pathological findings, thickness of the peritoneal membrane (P = 0.045), new membrane formation score (P = 0.006), ratio of luminal diameter to vessel diameter (L/V ratio, P<0.001), presence of CD31-negative vessels (P = 0.021), fibrin deposition (P<0.001), and collagen volume fraction (P = 0.018) were associated with EPS development. In analyses of samples with and without EPS matched for PD treatment period, non-diabetes, and PD solution, univariable analysis identified L/V ratio (per 0.1 increase: odds ratio (OR) 0.44, P = 0.003) and fibrin deposition (OR 6.35, P = 0.027) as the factors associated with EPS. L/V ratio was lower in patients with fibrin exudation than in patients without fibrin exudation.

Conclusions

These findings suggest that damage to vascular endothelial cells, as represented by low L/V ratio, could be a predictive finding for the development of EPS, particularly in long-term PD patients unaffected by peritonitis.

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<![CDATA[Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb9c

Introduction

Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions.

Methods

We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets.

Results

In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated.

Conclusions

Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.

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<![CDATA[Fibrin Sealants in Dura Sealing: A Systematic Literature Review]]> https://www.researchpad.co/article/5989d9deab0ee8fa60b689ab

Background

Fibrin sealants are widely used in neurosurgery to seal the suture line, provide watertight closure, and prevent cerebrospinal fluid leaks. The aim of this systematic review is to summarize the current efficacy and safety literature of fibrin sealants in dura sealing and the prevention/treatment of cerebrospinal fluid leaks.

Methods

A comprehensive electronic literature search was run in the following databases: Cochrane Database of Systematic Reviews, Cochrane Central Resister of Controlled Trials, clinicaltrials.gov, MEDLINE/PubMed, and EMBASE. Titles and abstracts of potential articles of interest were reviewed independently by 3 of the authors.

Results

A total of 1006 database records and additional records were identified. After screening for duplicates and relevance, a total of 78 articles were assessed by the investigators for eligibility. Thirty-eight were excluded and the full-text of 40 articles were included in the qualitative synthesis. Seven of these included only safety data and were included in the safety assessment. The remaining 33 articles included findings from 32 studies that enrolled a total of 2935 patients who were exposed to fibrin sealant. Among these 33 studies there were only 3 randomized controlled trials, with the remaining being prospective cohort analysis, case controlled studies, prospective or retrospective case series. One randomized controlled trial, with 89 patients exposed to fibrin sealant, found a greater rate of intraoperative watertight dura closure in the fibrin sealant group than the control group (92.1% versus 38.0%, p<0.001); however, post-operative cerebrospinal fluid leakage occurred in more fibrin sealant than control patients (6.7% versus 2.0%, p>0.05). Other clinical trials evaluated the effect of fibrin sealant in the postoperative prevention of cerebrospinal fluid leaks. These were generally lower level evidence studies (ie, not prospective, randomized, controlled trials) that were not designed or powered to demonstrate a significant advantage to fibrin sealant use. Two small case series studies evaluated the effect of fibrin sealants in persistent cerebrospinal fluid leak treatment, but did not establish firm efficacy conclusions. Specific adverse reports where fibrin sealants were used for dura sealing were limited, with only 8 cases reported in neurosurgical procedures since 1987 and most reporting only a speculative relationship/association with fibrin sealant exposure.

Conclusions

A major finding of this systematic literature review is that there is a paucity of randomized studies that have evaluated the effectiveness and safety of fibrin sealants in providing intraoperative watertight dura closure and post-operative cerebrospinal fluid leakage. Among the limited studies available, evidence from a single randomized, controlled trial indicates that fibrin sealants provide a higher rate of intraoperative watertight closure of the dura suture line than control, albeit with a higher rate of postoperative cerebrospinal fluid leakage. Evidence from non-randomized, controlled trials suggests that fibrin sealants may be effective in preventing cerebrospinal fluid leaks with an acceptable safety profile. There is a substantial need for randomized, controlled clinical trials or well-designed prospective observational trials where the conduct of a randomized trial is not feasible to fully assess the impact of fibrin sealant utilization on the rates of intraoperative dura closure, postoperative cerebrospinal leakage, and safety.

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<![CDATA[Untangling the Diverse and Redundant Mechanisms of Staphylococcus aureus Biofilm Formation]]> https://www.researchpad.co/article/5989da46ab0ee8fa60b8bc18 ]]> <![CDATA[A Combined In Vitro Imaging and Multi-Scale Modeling System for Studying the Role of Cell Matrix Interactions in Cutaneous Wound Healing]]> https://www.researchpad.co/article/5989d9e2ab0ee8fa60b6a063

Many cell types remodel the extracellular matrix of the tissues they inhabit in response to a wide range of environmental stimuli, including mechanical cues. Such is the case in dermal wound healing, where fibroblast migrate into and remodel the provisional fibrin matrix in a complex manner that depends in part on the local mechanical environment and the evolving multi-scale mechanical interactions of the system. In this study, we report on the development of an image-based multi-scale mechanical model that predicts the short-term (24 hours), structural reorganization of a fibrin gel by fibroblasts. These predictive models are based on an in vitro experimental system where clusters of fibroblasts (i.e., explants) were spatially arranged into a triangular geometry onto the surface of fibrin gels that were subjected to either Fixed or Free in-plane mechanical constraints. Experimentally, regional differences in short-term structural remodeling and cell migration were observed for the two gel boundary conditions. A pilot experiment indicated that these small differences in the short-term remodeling of the fibrin gel translate into substantial differences in long-term (4 weeks) remodeling, particularly in terms of collagen production. The multi-scale models were able to predict some regional differences in remodeling and qualitatively similar reorganization patterns for the two boundary conditions. However, other aspects of the model, such as the magnitudes and rates of deformation of gel, did not match the experiments. These discrepancies between model and experiment provide fertile ground for challenging model assumptions and devising new experiments to enhance our understanding of how this multi-scale system functions. These efforts will ultimately improve the predictions of the remodeling process, particularly as it relates to dermal wound healing and the reduction of patient scarring. Such models could be used to recommend patient-specific mechanical-based treatment dependent on parameters such as wound geometry, location, age, and health.

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<![CDATA[A Total Pleural Covering for Lymphangioleiomyomatosis Prevents Pneumothorax Recurrence]]> https://www.researchpad.co/article/5989dabdab0ee8fa60baf8df

Background

Spontaneous pneumothorax is a major and frequently recurrent complication of lymphangioleiomyomatosis (LAM). Despite the customary use of pleurodesis to manage pnenumothorax, the recurrence rate remains high, and accompanying pleural adhesions cause serious bleeding during subsequent lung transplantation. Therefore, we have developed a technique of total pleural covering (TPC) for LAM to wrap the entire visceral pleura with sheets of oxidized regenerated cellulose (ORC) mesh, thereby reinforcing the affected visceral pleura and preventing recurrence.

Methods

Since January 2003, TPC has been applied during video-assisted thoracoscopic surgery for the treatment of LAM. The medical records of LAM patients who had TPC since that time and until August 2014 are reviewed.

Results

TPC was performed in 43 LAM patients (54 hemithoraces), 11 of whom required TPC bilaterally. Pneumothorax recurred in 14 hemithoraces (25.9%) from 11 patients (25.6%) after TPC. Kaplan-Meier estimates of recurrence-free hemithorax were 80.8% at 2.5 years, 71.7% at 5 years, 71.7% at 7.5 years, and 61.4% at 9 years. The recurrence-free probability was significantly better when 10 or more sheets of ORC mesh were utilized for TPC (P = 0.0018). TPC significantly reduced the frequency of pneumothorax: 0.544 ± 0.606 episode/month (mean ± SD) before TPC vs. 0.008 ± 0.019 after TPC (P<0.0001). Grade IIIa postoperative complications were found in 13 TPC surgeries (24.1%).

Conclusions

TPC successfully prevented the recurrence of pneumothorax in LAM, was minimally invasive and rarely caused restrictive ventilatory impairment.

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<![CDATA[Influence of Interleukin-1 Beta on Platelet-Poor Plasma Clot Formation: A Potential Impact on Early Bone Healing]]> https://www.researchpad.co/article/5989da16ab0ee8fa60b7b311

Objectives

Hematoma quality (especially the fibrin matrix) plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1β) on fibrin clot formation from platelet-poor plasma (PPP).

Methods

Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG), blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10 min at 20°C. The effects of IL-1β cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM), confocal microscopy (CM), turbidity, and clot lysis assays.

Results

The lag time for protofibril formation was markedly shortened in the IL-1β treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1β and 97.5 ± 19.36 in the 500 pg/mL of IL-1β) compared to the control group without IL-1β (543.8 ± 205.8). Maximal turbidity was observed in the control group. IL-1β (500 pg/mL) treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05). The clot lysis assay revealed that 500 pg/mL IL-1β induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers.

Conclusion

IL-1β can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.

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<![CDATA[Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity]]> https://www.researchpad.co/article/5989da74ab0ee8fa60b96098

Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited ‘spontaneous’ plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.

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<![CDATA[PEGylation of Truncated Streptokinase Leads to Formulation of a Useful Drug with Ameliorated Attributes]]> https://www.researchpad.co/article/5989d9f0ab0ee8fa60b6e41b

Streptokinase (SK) remains a favored thrombolytic agent in the developing world as compared to the nearly 10-fold more expensive human tissue-plasminogen activator (tPA) for the dissolution of pathological fibrin clots in myocardial infarction. However, unlike the latter, SK induces systemic activation of plasmin which results in a greater risk of hemorrhage. Being of bacterial origin, it elicits generation of unwanted antibody and has a relatively short half-life in vivo that needs to be addressed to make it more efficacious clinically. In order to address these lacunae, in the present study we have incorporated cysteine residues specifically at the N- and C-termini of partially truncated SK and these were then PEGylated successfully. Some of the obtained derivatives displayed enhanced plasmin resistance, longer half-life (upto several hours), improved fibrin clot-specificity and reduced immune-reactivity as compared to the native SK (nSK). This paves the way for devising next-generation SK-based thrombolytic agent/s that besides being fibrin clot-specific are endowed with an improved efficacy by virtue of an extended in vivo half-life.

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<![CDATA[Extensive Characterization and Comparison of Endothelial Cells Derived from Dermis and Adipose Tissue: Potential Use in Tissue Engineering]]> https://www.researchpad.co/article/5989da76ab0ee8fa60b96c3c

Tissue-engineered constructs need to become quickly vascularized in order to ensure graft take. One way of achieving this is to incorporate endothelial cells (EC) into the construct. The adipose tissue stromal vascular fraction (adipose-SVF) might provide an alternative source for endothelial cells as adipose tissue can easily be obtained by liposuction. Since adipose-EC are now gaining more interest in tissue engineering, we aimed to extensively characterize endothelial cells from adipose tissue (adipose-EC) and compare them with endothelial cells from dermis (dermal-EC). The amount of endothelial cells before purification varied between 4–16% of the total stromal population. After MACS selection for CD31 positive cells, a >99% pure population of endothelial cells was obtained within two weeks of culture. Adipose- and dermal-EC expressed the typical endothelial markers PECAM-1, ICAM-1, Endoglin, VE-cadherin and VEGFR2 to a similar extent, with 80–99% of the cell population staining positive. With the exception of CXCR4, which was expressed on 29% of endothelial cells, all other chemokine receptors (CXCR1, 2, 3, and CCR2) were expressed on less than 5% of the endothelial cell populations. Adipose-EC proliferated similar to dermal-EC, but responded less to the mitogens bFGF and VEGF. A similar migration rate was found for both adipose-EC and dermal-EC in response to bFGF. Sprouting of adipose-EC and dermal-EC was induced by bFGF and VEGF in a 3D fibrin matrix. After stimulation of adipose-EC and dermal-EC with TNF-α an increased secretion was seen for PDGF-BB, but not uPA, PAI-1 or Angiopoietin-2. Furthermore, secretion of cytokines and chemokines (IL-6, CCL2, CCL5, CCL20, CXCL1, CXCL8 and CXCL10) was also upregulated by both adipose- and dermal-EC. The similar characteristics of adipose-EC compared to their dermal-derived counterpart make them particularly interesting for skin tissue engineering. In conclusion, we show here that adipose tissue provides for an excellent source of endothelial cells for tissue engineering purposes, since they are readily available, and easily isolated and amplified.

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<![CDATA[Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking]]> https://www.researchpad.co/article/5989da97ab0ee8fa60ba2496

The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis.

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<![CDATA[Thrombin Generation in Zebrafish Blood]]> https://www.researchpad.co/article/5989da8fab0ee8fa60b9f53e

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.

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<![CDATA[HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells]]> https://www.researchpad.co/article/5989db0bab0ee8fa60bca144

Background

During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis.

Objectives

We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling.

Methods and Results

Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction.

Conclusion

Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

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