Clot Structure Research Articles

Image-based analysis and simulation of the effect of platelet storage temperature on clot mechanics under uniaxial strain.

Optimal strength and stability of blood clots are keys to hemostasis and in prevention of hemorrhagic or thrombotic complications. Clots are biocomposite materials composed of fibrin network enmeshing platelets and other blood cells. We have previously shown that the storage temperature of platelets significantly impacts clot structure and stiffness. The objective of this work is to delineate the relationship between morphological characteristics and mechanical response of clot networks. We examined scanning electron microscope images of clots prepared from fresh apheresis platelets, and from apheresis platelets stored for 5days at room temperature or at 4°C, suspended in pooled plasma. Principal component analysis of nine different morphometric parameters revealed that a single principal component (PC1) can distinguish the effect of platelet storage on clot ultrastructure. Finite element analysis of clot response to uniaxial strain was used to map the spatially heterogeneous distribution of strain energy density for each clot. At modest deformations (25% strain), a single principal component (PC2) was able to predict these heterogeneities as quantified by variability in strain energy density distribution and in linear elastic stiffness, respectively. We have identified structural parameters that are primary regulators of stress distribution, and the observations provide insights into the importance of spatial heterogeneity on hemostasis and thrombosis.

The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood

Hypochlorite (HOCl), a strong oxidant and antimicrobial agent, has been proposed to be associated with hemostatic abnormalities during inflammatory response. However, its complex impact on hemostasis is not completely understood. In this report we studied the effect of clinically relevant (micromolar) HOCl concentrations on thrombus formation under flow, kinetics of platelet-fibrin clot formation, its architecture, retraction, and lysis. We found that HOCl (up to 500 µM) did not affect kinetics of coagulation measured in whole blood. HOCl (500-1000 µM) markedly diminished thrombus formation under flow. Clot retraction rate was reduced by HOCl dose-dependently (50-500 µM). HOCl (125-500 µM) inhibited fibrinolysis in whole blood and in platelet-depleted plasma, dose-dependently. Activity of plasmin was reduced by HOCl at concentrations started from 500 µM. HOCl (up to 500 µM) did not reduce plasminogen binding to fibrin under flow. HOCl (125-500 µM) modulated architecture of fibrin- and platelet-fibrin clots towards structures made of thin and densely packed fibers. Exposure of pure fibrinogen to HOCl (10-1000 µM) resulted in formation of dityrosine and was associated with altered fibrin structure derived from such modified fibrinogen. HOCl-altered fibrin net structure was not related with modulation of platelet procoagulant response, thrombin generation, and factor XIII activity. We conclude that, in human blood, clinically relevant HOCl concentrations may inhibit thrombus formation under flow, clot retraction and fibrinolysis. Fibrinolysis and clot retraction seem to be the most sensitive to HOCl-evoked inhibition. HOCl-modified fibrinogen and altered clot structure associated with it are likely to be primary sources of attenuated fibrinolysis.

Inflammatory cytokines in type 2 diabetes mellitus as facilitators of hypercoagulation and abnormal clot formation

BackgroundThe global burden of type 2 diabetes mellitus (T2DM), together with the presence of cardiovascular risk in this population, is reaching pandemic levels. A prominent feature of T2DM is chronic and systemic inflammation, with the accompanying presence of circulating and dysregulated inflammatory biomarkers; which in turn is associated with abnormal clot formation.MethodsHere, we investigate the correlation between abnormal blood clotting, using thromboelastography (TEG), clot ultrastructure using scanning electron microscopy (SEM) and the presence of a dysregulated inflammatory cytokine profile, by examining various circulating biomarkers.ResultsOur results show that many biomarkers, across TEG, cytokine and lipid groups, were greatly dysregulated in the T2DM sample. Furthermore, our T2DM sample’s coagulation profiles were significantly more hypercoagulable when compared to our heathy sample, and ultrastructural analysis confirmed a matted and denser clot structure in the T2DM sample.ConclusionsWe suggest that dysregulated circulating molecules may in part be responsible for a hypercoagulable state and vascular dysfunction in the T2DM sample. We propose further that a personalized approach could be of great value when planning treatment and tracking the patient health status after embarking on a treatment regimes, and that looking to novel inflammatory and vascular biomarkers might be crucial.

PB2432 POSSIBLE GENETIC RISK FACTORS OF PULMONARY EMBOLISM IN YOUNG PATIENTS FROM THE NORTH‐WESTERN REGION OF RUSSIA

Background:Pulmonary embolism (PE) and deep‐vein thrombosis (DVT) are the most common clinical manifestations of venous thromboembolism (VTE) which associated with high rates of morbidity and mortality in population. To date, little is known about the factors which could predict PE development in VTE patients. Several studies have shown that fibrin clot structure as well as the rate of clot lysis could influence the risk of PE development. Moreover, nucleotide variations in genes coding for components involved in processes of thrombus formation or its lysis are well‐described. Nevertheless, association between patient's genotype and features of clinical VTE manifestation, in particular, risk of PE development, remains debatable.Aims:To reveal possible association between the alpha‐fibrinogen (FI‐A), factor XIII A‐subunit (FXIII‐A), tissue plasminogen activator (TPA) and plasminogen activator inhibitor type I (PAI‐1) gene polymorphisms and the features of clinical VTE manifestation in patients with early‐onset disease from the North‐Western region of Russia.Methods:We analyzed 243 young patients under 45 years old (119 men and 124 women, mean age – 37.4 years) with at least one confirmed episode of acute VTE. Isolated DVT (“DVT only”) was diagnosed in 154 (63.4%), DVT complicated by PE (“DVT + PE”) – in 53 (21.8%), and isolated PE (“PE only”) – in 36 (14.8%) patients. Genotyping for the FI‐A Thr312Ala, FXIII‐A Val34Leu, PAI‐1 ‐675 4G/5G and TPA 311 bp Ins/Del variations was performed by PCR‐RFLP. To assess intergroup differences in genotype frequencies, the odds ratios (OR), their 95% confidence intervals (CI) and p‐values were calculated by Fisher's exact method.Results:Distributions of the FI‐A, FXIII‐A, PAI‐1 and TPA genotypes in patients with different clinical manifestations of VTE have shown several differences. Frequency of the TPA Del/Del genotype was significantly increased in VTE patients with the signs of PE when compared to those with isolated DVT (31.5% vs. 18.8%, respectively, OR = 2.0; 95% CI: 1.1‐3.6; p = 0.029). Homozygosity for the TPA Del allele was most prevalent in patients with isolated PE (38.9% vs. 18.8% in the group “DVT only”, OR = 2.7; 95% CI: 1.3‐6.0; p = 0.014). In this group, the frequency of the FI‐A 312 Ala/Ala variant was 2‐fold increased when compared to patients with isolated DVT (22.2% vs. 11.0%, respectively, OR = 2.3; 95% CI: 0.9‐5.9; p = 0.098). In patients with the signs of PE, homozygosity for the FXIII‐A 34Leu variant was observed more frequently than in the “DVT only” group (11.2% vs. 6.5%, respectively, OR = 1.8; 95% CI: 0.7‐4.6; p = 0.23). At the same time, the frequency of heterozygous FXIII‐A genotype was significantly increased in patients with isolated DVT when compared to “DVT + PE” group (48.7% vs. 30.2%, respectively, OR = 2.2; 95% CI: 1.1‐4.3; p = 0.024).Summary/Conclusion:TPA Del/Del genotype is associated with the risk of PE development in young VTE patients from the North‐Western region of Russia, whereas heterozygous FXIII‐A variant (34 Val/Leu) could protect from PE in patients with DVT. Role of the FI‐A 312 Ala/Ala and FXIII‐A 34 Leu/Leu variants as possible determinants of different clinical course of VTE needs further clarification.

Microfluidic and computational study of structural properties and resistance to flow of blood clots under arterial shear

The ability of a blood clot to modulate blood flow is determined by the clot’s resistance, which depends on its structural features. For a flow with arterial shear, we investigated the characteristic patterns relating to clot shape, size, and composition on the one hand, and its viscous resistance, intraclot axial flow velocity, and shear distributions on the other. We used microfluidic technology to measure the kinetics of platelet, thrombin, and fibrin accumulation at a thrombogenic surface coated with collagen and tissue factor (TF), the key clot-formation trigger. We subsequently utilized the obtained data to perform additional calibration and validation of a detailed computational fluid dynamics model of spatial clot growth under flow. We then ran model simulations to gain insights into the resistance of clots formed under our experimental conditions. We found that increased thrombogenic surface length and TF surface density enhanced the bulk thrombin and fibrin generation in a nonadditive, synergistic way. The height of the platelet deposition domain—and, therefore, clot occlusivity—was rather robust to thrombogenic surface length and TF density variations, but consistently increased with time. Clot viscous resistance was non-uniform and tended to be higher in the fibrin-rich, inner “core” region of the clot. Interestingly, despite intraclot structure and viscous resistance variations, intraclot flow velocity variations were minor compared to the abrupt decrease in flow velocity around the platelet deposition region. Our results shed new light on the connection between the structure of clots under arterial shear and spatiotemporal variations in their resistance to flow.

SUN-075 Higher Fibrinogen and Clot Density in Patients with Acromegaly: The Role of Adverse Body Composition to the Increased Thrombotic Potential

Introduction: Patients with acromegaly have increased mortality rates compared with the general population, particularly due to cardiovascular and cerebrovascular disease. We hypothesized that one mechanism through which GH/IGF-1 excess increases vascular disease is via effects on hemostasis/fibrinolysis and subsequent clot formation. Patients and Methods: We conducted a case-control study in 40 patients with acromegaly and 40 age and sex-matched healthy controls. We measured parameters of metabolic profile, fibrinogen using the Clauss method and performed clot structure analysis using a validated turbidimetric assay. Results: Forty patients with acromegaly (21 males, mean age 53±13 years) were recruited. 30% had active disease (GH>1mcg/L and IGF-1 above the reference range), 27.5% had dichotomous GH/IGF-1 results and 42.5% were in remission. Compared with controls, patients had a less favorable metabolic profile with higher BMI (30±5.5 vs. 26.7±4.1 kg/m2, p=0.003), waist/hip ratio (0.91±0.08 vs. 0.87±0.08, p=0.045), total fat mass (29.8±10 vs. 23.4±10 kg, p=0.003) and triglycerides (1.5±0.9 vs. 1.1±0.5 mmol/L, p=0.02). Significantly higher fibrinogen levels were found in the patient group (3.59±1.56 vs. 2.58±0.37 mg/ml, p<0.001). Patients had raised clot maximum absorbance (a measure of clot density), compared with controls (0.38±0.13 vs. 0.32±0.08 arbitrary units, p=0.02). Regarding fibrinolysis, no statistical difference in clot lysis times was found between the two groups, however patients with acromegaly had numerically more prolonged lysis times compared with controls at all lysis stages (times from maximum clot optic density to 25%, 50% and 75% lysis were respectively: 15.5±16.6 vs. 10.4±5.4 min, p=0.067; 29±22.2 vs. 23.2±11.8 min, p=0.15; and 40.8±22.5 vs. 35.3±12.5 min, p=0.18). In the patient group smoking was associated with higher fibrinogen levels (coefficient 0.8, p=0.03), higher clot density (coefficient 0.07, p=0.035) and reduced lag time for clot formation (coefficient -39.3, p=0.02). Diagnosis of diabetes, higher BMI and total fat mass were associated with more prolonged lysis time (coefficients 13, p=0.04; 1.7, p=0.007; and 0.9, p=0.009 respectively). Total fat mass was also positively associated with higher clot density (coefficient 0.005, p=0.01). Additionally, a positive correlation was found between fibrinogen and clot density (coefficient 0.05, p<0.001). Conclusions: Patients with acromegaly demonstrate abnormalities in clot structure properties, particularly in clot formation, with higher fibrinogen and clot density, suggesting increased thrombotic potential with more compact clots. The adverse metabolic profile of these patients is a contributing factor to these clotting abnormalities. Prospective studies are required to assess whether hemostatic abnormalities in acromegaly are reversible after successful treatment.

SUN-085 Assessing the Impact of Disease Activity on Clot Formation and Lysis in Patients with Acromegaly

Introduction: Data from previous studies suggest increased thrombogenic potential in patients with acromegaly, with higher levels of fibrinogen and a degree of impaired fibrinolysis, which may be contributing to the increased cardiovascular risk associated with acromegaly. However, it remains unclear whether these hemostatic abnormalities reverse in patients with disease remission. Patients and Methods: We conducted a cross-sectional study in 40 patients with acromegaly. The patients were divided into two groups based on the Endocrine Society criteria for disease remission (GH<1mcg/L and IGF-1 within the reference range) at the time of the study. We measured fibrinogen using the Clauss method and also analyzed clot formation and lysis using a validated turbidimetric assay. Results: Twenty-two patients with active acromegaly (Group 1; mean age 51±13 years) and 18 with disease remission (Group 2; mean age 55±13 years) were assessed. There was no difference in the two groups with regards to age, gender distribution, BMI, fat mass (as measured by bioimpedance), skinfold thickness, total cholesterol and HbA1c. Mean GH and IGF-1 values were significantly higher in the group with active acromegaly (2.6±2.4 vs. 0.5±0.5 mcg/L, p<0.001 and 163±105% vs. 73±23% of upper limit of normal, p<0.001 respectively). Mean duration of disease remission in Group 2 was 9.4±6 years. Patients in remission had more prolonged lag time for the initiation of clot formation (571±68 vs. 514±72 sec, p=0.02), however maximum clot optic density was similar in the two groups (Group 1: 0.40±0.12 vs. Group 2: 0.35±0.14 arbitrary units, p=0.26). Additionally, no statistical difference was found in the lysis times in the two groups (mean time from maximum clot formation to 50% lysis was 24.5±18.7 min in Group 1, compared with 34.4±25.3 min in Group 2; p=0.16). Fibrinogen levels were also similar (Group 1: 3.9±1.6 mg/ml vs. Group 2: 3.2±1.5 mg/ml; p=0.2). Duration of active disease was associated with higher fibrinogen levels (coefficient 0.06, p=0.03), while duration of disease remission was associated with longer lag time for clot formation (coefficient 5.0, p=0.015). Disease status was another predictor of lag time for clot formation, with active acromegaly being associated with shorter lag time (coefficient -56.5, p=0.016). Conclusions: We were not able to demonstrate any significant differences in the clot structure properties between patients with active acromegaly and those with disease remission. This may be due to a type II statistical error due to the relatively small sample size or suggest that hemostatic abnormalities described in other studies persist, despite biochemical control of acromegaly. The presence of active disease and the overall duration of this, appear to be associated with increased potential for clot formation in patients with acromegaly.

A direct oral anticoagulant edoxaban accelerated fibrinolysis via enhancement of plasmin generation in human plasma: dependent on thrombin-activatable fibrinolysis inhibitor

A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180ng/mL t-PA and 0.1nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.

The Effect of Exenatide on Cardiovascular Risk Markers in Women With Polycystic Ovary Syndrome.

Background: Polycystic ovary syndrome (PCOS) is associated with an adverse cardiovascular risk profile including a prothrombotic state. Exenatide has been shown to be effective at improving insulin sensitivity and weight loss in PCOS; therefore this study was undertaken to assess its effects on weight, endothelial function, inflammatory markers, and fibrin structure/function in overweight/obese women with PCOS.Methods: Thirty overweight/obese anovulatory women with all 3 Rotterdam criteria received exenatide 5 mcg bd for 4 weeks then 10 mcg bd for 12 weeks. The primary outcome was change in weight; secondary outcomes were changes in endothelial function [Reactive Hyperemia-Peripheral Arterial Tonometry (RH-PAT)], serum endothelial markers (ICAM-1, VCAM-1, E-selectin, and P-selectin), change in inflammation (hsCRP), and alteration in clot structure and function [maximum absorbance (MA), and time from full clot formation to 50% lysis (LT)].Results: Twenty patients completed the study. Exenatide reduced weight 111.8 ± 4.8 to 108.6 ± 4.6 kg p = 0.003. Serum endothelial markers changed with a reduction in ICAM-1 (247.2 ± 12.9 to 231.3 ± 11.5 ng/ml p = 0.02), p-selectin (101.1 ± 8.2 to 87.4 ± 6.6 ng/ml p = 0.01), and e-selectin (38.5 ± 3.3 to 33.6 ± 2.6 ng/ml p = 0.03), without an overt change in endothelial function. Inflammation improved (CRP; 8.5 ± 1.4 to 5.6 ± 0.8 mmol/L p = 0.001), there was a reduction in clot function (LT; 2,987 ± 494 to 1,926 ± 321 s p = 0.02) but not clot structure.Conclusion: Exenatide caused a 3% reduction in weight, improved serum markers of endothelial function, inflammation, and clot function reflecting an improvement in cardiovascular risk indices in these women with PCOS. This suggests exenatide could be an effective treatment for obese women with PCOS.Clinical Trial Registration: ISRCTN81902209.

Recurrent venous thromboembolism patients form clots with lower elastic modulus than those formed by patients with non‐recurrent disease

Essentials Venous thromboembolism (VTE) recurrence leads to decreased clot elastic modulus in plasma.Recurrent VTE is not linked to changes in clot structure, fiber radius, or factor XIII activity.Other plasma components may play a role in VTE recurrence.Prospective studies should resolve if clot stiffness can be used as predictor for recurrent VTE. SummaryBackgroundVenous thromboembolism (VTE) is associated with a high risk of recurrent events after withdrawal of anticoagulation.ObjectivesTo determine the difference in plasma clot mechanical properties between patients with recurrent VTE (rVTE) and those with non‐recurrent VTE (nrVTE).MethodsWe previously developed a system for determining clot mechanical properties by use of an in‐house magnetic tweezers system. This system was used to determine the mechanical properties of clots made from plasma of 11 patients with rVTE and 33 with nrVTE. Plasma was mixed with micrometer‐sized beads, and thrombin and calcium were added to induce clotting; the mixture was then placed in small capillary tubes, and clotting was allowed to proceed overnight. Bead displacements upon manipulation with magnetic forces were analyzed to determine clot elastic and viscous moduli. Fibrin clot structure was analyzed with turbidimetry and confocal microscopy. Factor XIII was measured by pentylamine incorporation into fibrin.ResultsClots from rVTE patients showed nearly two‐fold less elastic and less viscous moduli than clots from nrVTE patients, regardless of male sex, unprovoked events, family history of VTE, fibrinogen concentration, or body mass index. No differences were observed in clot structure, fibrinolysis rates, or FXIII levels.ConclusionUsing magnetic tweezers for the first time in patient samples, we found that plasma clots from rVTE patients showed a reduced elastic modulus and a reduced viscous modulus as compared with clots from nrVTE patients. These data indicate a possible role for fibrin clot viscoelastic properties in determining VTE recurrence.

DEVELOPMENT OF TECHNOLOGICAL SOUR-MILK DESSERTS ENRICHED WITH BIFIDOBACTERIA

The work is devoted to the development of the technology of fermented sour-milk desserts, enriched with bifidobacteria and biologically active and physiologically valuable substances of fruit-berry raw materials. Bifidobacteria regulate the quality and quantity composition of the normal intestinal microfllora that is an important factor of organism protection from different intestinal infections. The healthy effect is mainly conditioned by biologically valuable properties of specially selected consortiums of lacto- and bifidobacteria, more stable for the effect of inhibitors and unfavorable environmental conditions. At the joint use of selected consortiums of strains of bifido- and lactobacteria, the number of viable cells of bifidobacteria increases in 3-4 times, the antagonistic activity increases comparing with using separate strains of microorganisms. The use of fructose and lactulose as a biostimulating component of sour-milk desserts raises the activity, growth and development of bifidobacteria. For stabilizing the structure of sour-milk clots, there were used hydrocalloids, such as pectin and starch that favors the increase of the number of viable cells of bifidobacteria, provides the necessary viscosity and certain humidity, prevents stratification at using fruit-berry prebiotics. The optimal storage term of sour-milk desserts at temperature (3±1) оС is no more than 15 days

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications

AbstractBleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA–plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Fibrin Density and Tension Regulates Fibrinolytic Susceptibility

Fibrin fibers provide structural and mechanical integrity to blood clots until their dissolution during fibrinolysis. This work seeks to deconvolve the respective fibrinolytic influences of enzyme kinetics and enzyme perfusion rates into clots from clot structure and fiber properties. Here we consider the lysis of fibers within small, 2-D networks, which have polymerized between ridges. We tracked changes in fiber morphology within a 24-hour period during lysis. We find that fibers follow several possible degradation pathways during the lytic process including transverse cleavage, fiber bundling, structural elongation due to network rearrangements, tension loss, and network collapse. Transverse cleavage of individual fibers results in network rearrangements due to the redistribution of the tension in the yet-to-be-cleaved fibers. Network rearrangements and remnants of cleaved fibers often lead to fibers bundling into thicker fibers which are more resistant to fibrinolysis. When the final strand holding the network in place is transversely cleaved, the remaining fibers recoil and collapse onto the ridge. These effects highlight the inherent tension in fibrin networks and its role in governing fibrinolytic degradation. This is further emphasized by the observation that lysis kinetics depend strongly on fiber density. Fibers in high-density areas, and presumably under more tension, lyse at earlier time points and more rapidly than fibers in low-density areas. Fibrinolysis initiated with low (0.1 U/mL) concentrations of plasmin occurs more slowly than with high (1.0 U/mL) concentrations of plasmin, and structural rearrangement due to tension redistribution is more prominent. These results identify the different pathways whereby a fibrin network is cleared out of an area by the lytic enzyme plasmin. Inherent fiber tension plays an important role in regulating this process.

Altered fibrin clot properties in advanced lung cancer: impact of chemotherapy.

Faster formation of dense and poorly lyzable fibrin networks have been reported in patients at risk of thromboembolism, including cancer patients. We sought to investigate whether chemotherapy affects plasma fibrin clot properties and their determinants in lung cancer patients. In this observational study we enrolled 83 consecutive patients with advanced inoperable lung cancer. Plasma fibrin clot permeability (Ks), turbidimetric analysis of clot formation, clot lysis time (CLT), microparticle-associated tissue factor (MP-TF) activity, and thrombin generation parameters were investigated at enrolment and 3-4 months after standard chemotherapy. Lung cancer patients after 4 (range, 4-5) cycles of chemotherapy had 35.6% higher D-dimer, 22.1% lower MP-TF activity, and unaltered fibrinogen compared with baseline. Chemotherapy resulted also in 7.5% increased Ks, 8.6% prolonged lag phase, and 5.4% shortened CLT, while thrombin generation was unchanged. Chemotherapy-related differences in clot structure were confirmed by scanning electron microscopy images. Fibrin clot properties after chemotherapy did not differ among histological types of lung cancer, cancer stages or chemotherapy regimens. Interestingly, never smoking (n=13, 16%) was associated with looser post-treatment fibrin structure as reflected by 12.3% higher Ks. Multiple linear regression showed that more advanced cancer stage, higher peak thrombin generation, and higher white blood cell count determined post-treatment change in Ks, while active smoking was associated with change in CLT. Three-month chemotherapy in lung cancer patients improves clot properties despite unaffected thrombin generation, suggesting that anticancer treatment might quickly produce antithrombotic actions.

Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (Aβ) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). Aβ, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This Aβ-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. Aβ-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the Aβ-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of Aβ and Aβ-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of Aβ-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate Aβ-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified Aβ-fibrinogen interaction inhibitor.

Analysis of &amp;#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of &amp;#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Intravascular Cells and Circulating Microparticles Induce Procoagulant Activity Via Phosphatidylserine Exposure in Venous Thromboembolism and Contribute to Altered Plasma Fibrin Clot Properties

Introduction:Venous thromboembolism(VTE), encompassing pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major contributor to global morbidity and mortality, especially among cancer patients. However, the factors that contribute to PE, a prevalent and fatal complication of DVT, remain poorly understood.Despite a common pathology relating PE to DVT, this leaves a grey area in the differences among DVT, confirmed PE, or DVT with PE, and the definitive relationship between VTE and this hypercoagulable state.However, relatively little is known about the definitive role of phosphatidylserine (PS), one of the blood constituents in the coagulant pathway, in the hypercoagulability of VTE and their altered plasma fibrin clot properties. Our objectives were to assess the levels of PS exposure on microparticles (MPs), blood cells, endothelial cells(ECs) and cell-derived MPs in each group of VTE and to evaluate their procoagulant activity (PCA), as well as the correlation between initial PS exposure and their altered plasma fibrin clot properties.Methods:DVT alone (n=27), definitely PE alone (n=22), DVT with PE (n=19) and healthy controls (n=30) were enrolled in the present study. PS exposure on MPs, blood cells and cultured ECs treated with VTE serum in vitro was analyzed with flow cytometry and confocal microscopy. MPs were classified based on their cellular origin: platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), endothelial cells(CD31+CD41a-, EMPs), erythrocytes (CD235a+, RMPs), monocytes (CD14+, MMPs), T lymphocytes (CD3+, T LyMPs), and B lymphocytes (CD19+, B LyMPs). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+MPs, blood cells and ECs were performed by lactadherin. Abnormal fibrin clot properties measured ex vivo in plasma was examined by fibrin clot analysis and scanning electron microscopy (SEM).Results: There was no significant difference in MP cellular origin between healthy and VTE subjects. However, the total number of PS+MPs was significantly increased (P < 0.001 for total MP and all MP subtypes) in VTE patients compared with healthy controls. In addition, circulating PS+ MPs cooperated with PS+blood cells and cultured ECs to markedly shorten coagulation time (Figure 1A) and dramatically increase FXa/thrombin generation and fibrin formation in each VTE group (all P < 0.05). Confocal microscopy images showed that the FVa/FXa complex and fibrin strands co-stained with PS on ECs. Moreover, blockade of exposed PS on MPs and intravascular cells with lactadherin inhibited PCA by approximately 80% (Figure 1B). Fibrin clot structure was evaluated in 15 VTE patients (DVT=5, PE=5 and DVT with PE=5) and 5 healthy subjects.Compared with those from DVT and PE, clots from plasma of DVT with PE subjects showed lower fiber density and faster clot lysis time, whereas there were no differences in lag time, rate of clot formation and maximum absorbance of turbidity.Conclusions: Our results suggest that compared with healthy subjects, each group of VTE patients had markedly higher levels of circulating PS+MPs, PS+blood cells. Additional, ECs treated with VTE patient serum had elevated percentages of PS+cells versus those treated with serum from healthy controls. Blockade of PS with lactadherin significantly inhibits PCA of blood cells, MPs and VTE serum-treated ECs. In addition, differences in fibrin clot properties and clot structure among DVT, PE and DVT with PE subjects may provide important insights into the pivotal mechanisms that regulate embolization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Combined Effect of By-Pass Agents and a Sequence Identical Analogue of Emicizumab on Fibrin Clot Turbidity and Structure in Factor VIII Deficient Plasma

Development of inhibitors to Factor VIII is a serious and not uncommon (occurs in about 30% of treated patients) complication of the treatment with factor concentrate in hemophilia A. Treatment of patients with inhibitors is complicated, expensive and not always successful. By-pass agents (rFVIIa and aPCC) have been considered as a treatment of choice for those patients. Very recently bispecific factor IXa- and factor X-directed antibody - Emicizumab has been approved for the treatment of hemophilia A patients with inhibitors. However, there are data from the clinical studies which raise concerns about potential prothrombotic effect of Emicizumab in combination with by-pass agents particularly aPCC.To elucidate a potential mechanism of hypercoagulability we have investigated fibrin clot quality in hemophilic plasma after addition of a sequence identical analogue (SIA) of Emicizumab in combination with by-pass agents.Parameters of fibrin clot turbidity (lag time, Max Abs, Slope, Slope time) were measured in recalcified FVIII-deficient plasma samples, after addition of low thrombin concentration (0.04 NIH/mL) and different concentrations of SIA (200 and 600 nM), rFVIIa (1.75 and 5.25 µg/mL) and aPCC (50 and 500 mU/mL). Pooled normal plasma (PNP) was used as control.After fixation, fibrin clots were analyzed by scanning electron microscopy (SEM) (Carl Zeiss, Oberkochen, Germany) and the thickness of individual fibers was measured using SIS iTEM software (FEI Company, Eindhoven, Netherlands). As previously determined, 50 randomly selected fibers in each sample were measured to obtain stable mean.All parameters of fibrin turbidity and structure were analyzed in triplicate and the results are presented as mean values.Fibrin clot in FVIII deficient plasma was difficult to analyze due to the lack of polymerization of fibrin monomers and absence of fiber formation. This structure is loose and prone to fibrinolysis. Interestingly the similar structure was observed after addition of both concentrations of rFVIIa while after addition of SIA fibrin structure improves. The best effect using single agent was noticed after addition of aPCC in concentration of 500 mU/mL. In combination with SIA (both concentrations of 200 nM and 600 nM) aPCC in concentrations of 500 mU/mL generated clots with highest density formed from very thin fibers and small intrinsic pores. These clots are even tighter than those formed from PNP samples.Parameters of fibrin turbidity obtained with higher concentrations of agents as well as representative SEM scans are presented on the Figure 1.Addition of SIA to by-pass agents additionally improved fibrin quality in FVIII deficient plasma. Combination of therapeutic concentrations of aPCC and SIA may produce clots even less porous than those in normal plasma. [Display omitted] DisclosuresChaireti:Shire: Research Funding. Antovic:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Werfen: Honoraria; Stago: Honoraria; Siemens: Honoraria; Roche: Honoraria; Sysmex: Honoraria.

Computational Modeling and Microfluidics Reveal Characteristic Patterns of Regulation of Clot Structure and Mechanics By Tissue Factor Localization

The growth of a blood clot is initiated by tissue factor (TF) exposure and is expected to depend on TF localization (i.e., its level and spatial distribution). We sought to understand how the structural and mechanical properties of clots under flow are shaped by simultaneously varying the surface density of TF and its area of exposure. We used a microfluidic device harboring thrombogenic surfaces that differed in length and TF surface density. We perfused human whole blood through this device and continually measured platelet deposition, thrombin formation, and fibrin accumulation by means of fluorescence microscopy. Using our recently developed, detailed mathematical model of spatial clot growth under flow, we performed computational simulations to gain insights into the connection between the structure of a clot and its mechanical properties, such as resistance to blood flow and flow axial velocity.We detected a non-additive, synergistic influence of the thrombogenic surface length and TF surface density on bulk thrombin and fibrin generation. We found that thrombogenic surface length and TF density controlled not only bulk accumulation, but also the occlusivity of the deposited platelet mass, as well as clot resistance to flow. The extent of this control depended on the blood flow velocity. An increase in the TF surface density resulted in condition-dependent differential acceleration of platelet deposition, thrombin formation, and fibrin accumulation. The viscous resistance of the clot was characterized by spatial variations and was higher in the inner clot region. Notably, despite variations in the intraclot structure, variations in the intraclot flow velocity were minor compared to the abrupt decrease in flow velocity at the boundary of the platelet deposition domain.Our analysis revealed characteristic patterns that describe how the shape, size, internal structure, and viscous resistance of a clot depend on the surface density of TF and its area of exposure. Taken together, our results suggest that the structure and mechanics of a growing clot are correlated, but can differ in their regulation by the distinct aspects of TF localization. These findings provide new insights into how initiating signals can temporally and spatially affect thrombus growth under flow. DISCLAIMERThe opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Army or of the U.S. Department of Defense. This abstract has been approved for public release with unlimited distribution. DisclosuresNo relevant conflicts of interest to declare.