Plasma Clot Formation Research Articles

One-Pot Fabrication of Highly Flexible Fluorine-Free Lubricant-Infused Poly(vinyl alcohol) Films with Superior Antifouling Properties.

In clinical settings, biofluid-contacting devices can suffer from biofouling, leading to thrombus formation and bacterial biofilm buildup, which impair device function and pose health risks. Traditional antifouling methods, including the use of hydrophilic polymers and heparin coatings, often suffer from instability and reduced bioactivity over time. Lubricant-infused surfaces (LIS) have emerged as a promising alternative due to their long-term stability and broad-spectrum repellency. However, current LIS technologies typically involve complex, multistep processes that restrict their application to surface layers, potentially compromising performance under mechanical stress. This study introduces a novel method for bulk modification of poly(vinyl alcohol) (PVA) films, creating flexible lubricant-infused PVA membranes with superior antifouling properties. These films are fabricated by cross-linking the PVA chains using n-propyltrichlorosilane (n-PTCS) and subsequent infusion with silicone oil as a lubricant. The modified PVA films significantly prevent bacterial adhesion and prolong blood and plasma clot formation. Additionally, these films exhibit enhanced mechanical properties, particularly in elasticity and flexibility compared to unmodified PVA films. The developed technique provides a straightforward method for creating flexible, super-repellent biointerfaces with the potential to prevent blood adhesion and bacterial biofilm formation, which are common complications associated with biofluid-contacting devices and medical implants.

Hemocompatibility of Albumin-Modified Magnetic Nanoparticles.

Kidney failure leads to the accumulation of metabolites in the blood compartment. This build-up of metabolites has been associated with increased mortality and morbidity in these patients; thus, these metabolites are commonly called uremic toxins. The retention of some uremic toxins in the blood results from a strong interaction with serum albumin, preventing their clearance using standard hemodialysis techniques. Adsorbents are considered the next-generation technology for clearing uremic toxins from the blood, and iron oxide magnetic nanoparticles are a promising material due to a high surface area that is easily modified and the ability to remove them from blood with an external magnetic field. Plasma protein adsorption and clot formation kinetics were determined for unmodified and albumin-modified iron oxide magnetic nanoparticles. Albumin was selected because it can bind uremic toxins, and it is commonly used to passivate surfaces. Coatings were formed and characterized using transmission electron microscopy, thermogravimetric analysis, and zeta-potential analysis. Clotting kinetics, total protein assays, and immunoblots were used to analyze the effect surface modification has on protein adsorption events. Unmodified nanoparticles showed rapid clotting and more adsorbed protein compared to albumin-coated iron oxide nanoparticles. Immunoblots show that modified particles showed changes in albumin, protein C, Immunoglobulin G, transferrin, fibrinogen, α1-antitrypsin, vitronectin, plasminogen, prothrombin, and antithrombin levels compared to unmodified controls. The hemocompatibility of adsorbent materials is essential to their clinical application in clearing the blood of uremic toxins.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release invivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation invitro (P<0.01). We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Determination of thrombin and plasmin activity using the turbidimetric analysis of clot formation and dissolution in human blood plasma

Based on the turbidimetric curve of formation and dissolution of a blood plasma clot initiated by the activated partial thromboplastin time reagent, a method for determining the coagulation component of thrombin activity and fibrinolytic activity of plasmin is proposed. The activity of thrombin was calculated by the value of the lag period, and plasmin by its amidase activity at the moment of complete dissolution of the clot. At the end of the lag period, about 0.45% of the available prothrombin was activated, and at the moment of complete dissolution of the clot 1.05% of the available plasminogen was activated. This method makes it possible to determine the ratio of the thrombin generation rate to that of plasmin, the time of clot formation to the time of its dissolution, as well as the overall hemostasis potential and coagulation and fibrinolytic components and their ratio. Keywords: coagulation, fibrinolysis, global hemostasis assay, plasmin generation, thrombin generation

Plasma Clot Properties in Patients with Pancreatic Cancer.

Pancreatic cancer is one of the most prothrombotic malignancies. Plasma clot properties may be altered in patients with pancreatic cancer, and circulating tissue factor (TF) may play an important role. We applied a modified plasma clot formation assay (only CaCl2 and phospholipids were added to initiate clotting) and a standard clotting assay (lipidated TF was also added) to investigate whether plasma clot properties are altered in pancreatic cancer patients (n = 40, 23 female) compared to sex-matched healthy controls. The modified assay was also performed in the presence of a TF blocking antibody. With this modified assay, we detected an increased plasma clot formation rate (Vmax) and an increased delta absorbance (ΔAbs, indicating fibrin fiber thickness) in patients compared to controls. These differences were not detected with the standard clotting assay. Following addition of a TF blocking antibody in in our modified assay, Vmax decreased significantly in patients only, ΔAbs significantly decreased in patients and in healthy controls, the lag phase did not change, and the time to peak fibrin generation increased in patients only. Taken together, these findings indicate the presence of a prothrombotic state in pancreatic cancer patients, which depends on TF and is detectable with our modified assay but not with a standard clotting assay.

Super-Repellent and Flexible Lubricant-Infused Bacterial Nanocellulose Membranes with Superior Antithrombotic, Antibacterial, and Fatigue Resistance Properties.

Bacterial nanocellulose (BNC) is a naturally derived hydrogel that has recently paved its way in several biomedical applications. Despite its remarkable tissue-like properties, BNC does not express innate anticoagulant or antimicrobial properties; therefore, appropriate post-modification procedures are required to prevent nonspecific adhesion and enhance the hemocompatibility properties of BNC-based biointerface. Here, we report a new class of flexible, lubricant-infused BNC membranes with superior antithrombotic and antibacterial properties. Using chemical vapor deposition, porous BNC membranes were functionalized with fluorosilane molecules and further impregnated with a fluorocarbon-based lubricant. Compared with unmodified BNC membranes and commercially available poly(tetrafluoroethylene) (PTFE) felts, our developed lubricant-infused BNC samples significantly attenuated plasma and blood clot formation, and prevented bacterial migration, adhesion, and biofilm formation and exhibited superior fat and enzyme repellency properties. Moreover, when subjected to mechanical testing, the lubricant-infused BNC membranes demonstrated a significantly higher tensile strength and greater fatigue resistance when compared with unmodified BNC samples and PTFE felts. Overall, the superior mechanical strength and antithrombotic, antibacterial, and fat/enzyme resistant properties observed in the developed super-repellent BNC-based membranes render their application promising for various biofluid-contacting medical implants and tissue engineering constructs.

Tissue factor pathway inhibitor is a potential modifier of bleeding risk in factor XI deficiency

BackgroundFactor (F) XI deficiency is associated with increased bleeding risk in some individuals. Neither FXI levels nor clinical clotting assays predict the bleeding risk. Compared with controls, FXI-deficient bleeders have reduced clot formation, decreased fibrin network density, and increased susceptibility to fibrinolysis. Tissue factor pathway inhibitor (TFPI) was recently implicated as a modifying factor in individuals with bleeding of unknown cause. ObjectivesTo determine the potential of TFPI in modifying the bleeding risk in FXI-deficient individuals. MethodsThe effects of TFPI on thrombin generation and clot formation, structure, and fibrinolysis in FXI-deficient plasma were measured in vitro in the absence or presence of inhibitory anti-TFPI antibody or exogenous recombinant TFPIα. Total plasma TFPI concentration was measured in 2 independent cohorts of controls and FXI-deficient individuals classified as bleeders or nonbleeders (cohort 1: 10 controls and 16 FXI-deficient individuals; cohort 2: 48 controls and 57 FXI-deficient individuals) and correlated with ex vivo plasma clot formation and fibrinolysis parameters associated with bleeding risk. ResultsIn an in vitro FXI deficiency model, inhibition of TFPI enhanced thrombin generation and clot formation, increased the network density, and decreased fibrinolysis, whereas an increase in TFPI had the opposite effects. Compared with controls, plasma from FXI-deficient bleeders had higher TFPI concentration. Total plasma TFPI concentrations correlated with parameters from ex vivo clotting and fibrinolysis assays that differentiate FXI-deficient bleeders and nonbleeders. ConclusionCoagulation and fibrinolysis parameters that differentiate FXI-deficient nonbleeders and bleeders were altered by plasma TFPIα. Total plasma TFPI was increased in FXI-deficient bleeders. TFPI may modify the bleeding risk in FXI-deficient individuals.

Cancer Cells and Platelets Have a Synergistic Role on the Acceleration of Clot Formation and Fibrin Polymerization. a Thromboelastometric Study

Introduction The histological type and the aggressiveness of cancer cells as well as the acquisition of resistance to chemotherapy are significant predictors for the high risk of cancer associated thrombosis (CAT). It is well established that cancer cells, by expressing tissue factor (TF) trigger thrombin generation. Cancer cell derived extracellular vesicles (CaCa-dEV) bearing TF, in the microenvironment, further enhance thrombin generation. The impact of cancer cells on clot formation kinetics and the qualitative characteristics of the clot has not been evaluated yet. Aim The present study aimed to analyze the variables which influence the kinetics and the qualitative characteristics of the clot after triggering thrombin generation by cells from pancreatic cancer (BXPC3), breast cancer (MCF7 and MCF7R10) and multiple myeloma (RPMI-8226 and U266) - associated with different risk of CAT. The impact of the resistance to chemotherapy on the clot characteristics was also studied. Methods Cancer cell lines (BXPC3, MCF7, MCF7R10, RPMI-8226 and U266) and primary umbilical vein endothelial cells (HUVEC) for the control experiments were used according to standardized and validated experimental procedure. Doxorubicin resistant breast cancer cells (MCF7R10) were obtained by treating native breast cancer cells (MCF7) by increasing concentrations of Doxorubicin from 5 nM to 10 µM.. Thromboelastometry, using the ROTEM® delta device (from Warfen Paris, France), was used for the study of clot formation and firmness in whole blood (WB), platelet rich and platelet poor plasma (PRP and PPP respectively) in the presence of cancer cells or HUVEC (control). A suspension of 200 μL with 105 cells was placed in each sterilized cup. Following an incubation of 4 hour, culture media was removed and 330 μL of WB, PRP or PPP were added followed by addition of 20 µM of CaCl2 (0.2 M). Clotting time (CT), α-angle and maximum clot firmness (MCF) were analyzed. Results In PPP, pancreatic cancer cells BXPC3 as well as chemotherapy sensitive and resistant breast cancer cells (MCF7 and MCF7R10), significantly reduced the CT. MCF7R10 cells significantly reduced the CT as compared to MCF7. In contrast, myeloma cells (RPMI8226 and U266), did not significantly affect the CT. All studied cancer cell lines significantly increased the α-angle as compared to HUVEC, with the most potent effect from BXPC3 cells. The studied cancer cell lines did not significantly affect MCF. In PRP, BXPC3 cells significantly reduced the CT whereas MCF7 and MCF7R10 did not significantly change the CT, and myeloma cells significantly prolonged the CT as compared to HUVEC. BXPC3 cells significantly increased the α-angle as compared to the HUVEC, whereas the other studied cancer cell lines did not have any significant effect on this parameter. BXPC3, MCF7 and MCF7R10 cells did not significantly affect the MCF whereas the myeloma cells significantly decreased the MCF as compared to HUVEC. In WB, BXPC3 and MCF7 cells significantly decreased the CT whereas MCF7R10 did not show any significant effect and the myeloma cells significantly prolonged it. The α-angle significantly increased in the presence of BXPC3 cells whereas the other cell lines did not show any significant effect on this parameter. The presence of cancer cells resulted in significant decrease of the MCF as compared to the control (HUVEC), with the most potent effect from myeloma. In the control experiment (HUVEC), the comparison of the thromboelastometric profile of the three experimental systems (PPP, PRP and WB) showed that platelets (PRP) had a substantial impact on CT, α-angle and MCF whereas the erythrocytes (in WB) did not further modify these parameters. The same pattern of the role of platelets was observed in the presence of the studied cancer cell lines. Discussion The present study provides experimental evidence documenting that cancer cells impact clot formation kinetics without affecting clot firmness. The impact of cancer cells on clot formation kinetics is determined principally by the histological type. The pancreatic adenocarcinoma cells have a more pronounced effect on clot formation kinetics as compared to the breast cancer cells (sensitive or resistant to Doxorubicine). In contrast, multiple myeloma cells delay fibrin formation and polymerization and decrease the clot firmness. Platelets have a synergistic effect with cancer cells on the acceleration of the clot formation kinetics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hyperfibrinolysis drives mechanical instabilities in a simulated model of trauma induced coagulopathy

IntroductionTrauma induced coagulopathy (TIC) is common after severe trauma, increasing transfusion requirements and mortality among patients. TIC has several phenotypes, with primary hyperfibrinolysis being among the most lethal. We aimed to investigate the contribution of hypercoagulation, hemodilution, and fibrinolytic activation to the hyperfibrinolytic phenotype of TIC, by examining fibrin formation in a plasma-based model of TIC. We hypothesized that instabilities arising from TIC will be due primarily to increased fibrinolytic activation rather than hemodilution or tissue factor (TF) induced hypercoagulation. MethodsThe influence of TF, hemodilution, fibrinogen consumption, tissue plasminogen activator (tPA), and the antifibrinolytic tranexamic acid (TXA) on plasma clot formation and structure were examined using rheometry, optical properties, and confocal microscopy. These were then compared to plasma samples from trauma patients at risk of developing TIC. ResultsCombining TF-induced clot formation, 15 % hemodilution, fibrinogen consumption, and tPA-induced fibrinolysis, the clot characteristics and hyperfibrinolysis were consistent with primary hyperfibrinolysis. TF primarily increased fibrin polymerization rates and reduced fiber length. Hemodilution decreased clot optical density but had no significant effect on mechanical clot stiffness. TPA addition induced primary clot lysis as observed mechanically and optically. TXA restored mechanical clot formation but did not restore clot structure to control levels. Patients at risk of TIC showed increased clot formation, and lysis like that of our simulated model. ConclusionsThis simulated TIC plasma model demonstrated that fibrinolytic activation is a primary driver of instability during TIC and that clot mechanics can be restored, but clot structure remains altered with TXA treatment.

Blocking domain 6 of high molecular weight kininogen to understand intrinsic clotting mechanisms

BackgroundThe contact system is initiated by factor (F) XII activation and the assembly of high molecular weight kininogen (HK) with either FXI or prekallikrein (PK) on a negatively charged surface. Overactivation ofsystem contributes to thrombosis and inflammation in numerous diseases. To develop effective therapeutics for contact system disorders, a detailed understanding ofpathway is needed. MethodsWe performed coagulation assays in normal human plasma and various factor‐deficient plasmas. To evaluate how HK‐mediated PK and FXI activation contributes to coagulation, we used an anti‐HK antibody to block access to domain 6 of HK, the region required for efficient activation of PK and FXI. ResultsFXI's binding to HK and its subsequent activation by activated FXII contributes to coagulation. We found that the 3E8 anti‐HK antibody can inhibit the binding of FXI or PK to HK, delaying clot formation in human plasma. Our data show that in the absence of FXI, however, PK can substitute for FXI inprocess. Addition of activated FXI (FXIa) or activated PK (PKa) abolished the inhibitory effect of 3E8. Moreover, the requirement of HK in intrinsic coagulation can be largely bypassed by adding FXIa. Like FXIa, exogenous PKa shortened the clotting time in HK‐deficient plasma, which was not due to feedback activation of FXII. Conclusionsstudy improves our understanding of HK‐mediated coagulation and provides an explanation for the absence of bleeding in HK‐deficient individuals. 3E8 specifically prevented HK‐mediated FXI activation; therefore, it could be used to prevent contact activation‐mediated thrombosis without altering hemostasis.

Chronic spontaneous urticaria: a low-grade disseminated intravascular coagulation only partially reversed by Omalizumab

Chronic spontaneous urticaria (CSU) is a disorder characterized by wheals and/or angioedema. The coagulation cascade and inflammation pathways are closely linked together. The aim of our study was first to investigate the dynamics of clot formation in plasma (Clot Waveform Analysis, CWA) in a group of 47 patients with CSU along with other coagulative parameters dedicated to the study of hypercoagulability, such as D-Dimer, F 1 + 2 peptide, Fibrinogen, Platelet count and Mean Platelet Volume (MPV). Secondly, 23 out of 47 patients were treated with Omalizumab at four administration intervals from T0 to T4. A statistically significant increase in Activated Partial Thromboplastin (aPTT) ratio, D-Dimer, F1 + 2, Platelet count and MPV was found when compared with 53 healthy controls (HC). In contrast, the 2nd Derivative of aPTT showed lower values than those of the HC. No differences were found between 1st derivative of aPTT and Fibrinogen. D-Dimer only showed a significant difference between T0 and T3. An activation of both coagulation and fibrinolysis along with a weaker clot acceleration may be in agreement with a low-grade DIC. The accelerated turnover of platelets expressed by both an increase in platelet count and MPV further supports this pathway in CSU. Omalizumab does not affect the relationship between the immune and the hemostatic systems.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Both G protein–coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice

AbstractPlatelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein–coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet–leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet–neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Thrombomodulin in patients with mild to moderate bleeding tendency.

IntroductionA massive increase of soluble thrombomodulin (sTM) due to variants in the thrombomodulin gene (THBD) has recently been identified as a novel bleeding disorder.AimTo investigate sTM levels and underlying genetic variants as a cause for haemostatic impairment and bleeding in a large number of patients with a mild to moderate bleeding disorder (MBD), including patients with bleeding of unknown cause (BUC).Patients and methodsIn 507 MBD patients, sTM levels, thrombin generation and plasma clot formation were measured and compared to 90 age‐ and sex‐matched healthy controls. In patients, genetic analysis of the THBD gene was performed.ResultsNo difference in sTM levels between patients and controls was found overall (median ([IQR] 5.0 [3.8‐6.3] vs. 5.1 [3.7‐6.4] ng/ml, p = .762), and according to specific diagnoses of MBD or BUC, and high sTM levels (≥95th percentile of healthy controls) were not overrepresented in patients. Soluble TM levels had no impact on bleeding severity or global tests of haemostasis, including thrombin generation or plasma clot formation. In the THBD gene, no known pathogenic or novel disease‐causing variants affecting sTM plasma levels were identified in our patient cohort.ConclusionTM‐associated coagulopathy appears to be rare, as it was not identified in our large cohort of patients with MBD. Soluble TM did not arise as a risk factor for bleeding or altered haemostasis in these patients.

TLR3 promotes venous thrombosis through neutrophil recruitment

Venous thromboembolism (VTE) remains the third most common cause of mortality among all cardiovascular diseases. Innate inflammatory mechanisms contribute to the pathogenesis of VTE. Toll-like receptors (TLRs) play pivotal roles in the innate immune system by detecting “danger signals”. Endogenous RNA has been identified as a potent procoagulant factor in thrombosis and may possibly act through TLR3. To investigate how eRNA mediate procoagulant effects through endothelial TLR3 in venous thrombosis. The FeCl 3 model was used to induce venous thrombosis in WT or TLR3 deficient (-/-) mice. A specific fluorescent probe for RNA (Syto RNA Select), RNase1, poly(I:C) or RNA extracted from murine endothelial cells (eRNA) were injected to mice. HUVECs were treated with poly(I:C) or eRNA extracted from HEK 293 cells in the presence of a TLR3 antagonist. Gene expression and signaling pathways activated were analyzed using western blot and real-time quantitative PCR. Turbidimetric measurement of plasma clot formation was performed by incubating HUVECs treated with poly(I:C) or eRNA with human plasma. We found that FeCl 3 promoted RNA release in vivo and increased the RNA content within the thrombus. RNase1 treatment reduced thrombus size compared to control mice. In contrast, eRNA and poly(I:C) treatment increased thrombus size in WT mice whereas no modifications were observed in TLR3-/- mice. Hence, poly(I:C) and eRNA treatments bolstered neutrophil infiltration in WT but not in TLR3-/- mice. In vitro, eRNA stimulated the release of CXCL5 in WT but not in TLR3-/- endothelial cells. In addition, eRNA enhanced plasma clot formation by downregulating thrombomodulin mRNA expression. These effects are mediated through NFkB activation. These results suggest that eRNA through TLR3 enhances neutrophil recruitment and clot formation leading to venous thrombosis.

Polysiloxane Nanofilaments Infused with Silicone Oil Prevent Bacterial Adhesion and Suppress Thrombosis on Intranasal Splints.

Like all biofluid-contacting medical devices, intranasal splints are highly prone to bacterial adhesion and clot formation. Despite their widespread use and the numerous complications associated with infected splints, limited success has been achieved in advancing their safety and surface biocompatibility, and, to date, no surface-coating strategy has been proposed to simultaneously enhance the antithrombogenicity and bacterial repellency of intranasal splints. Herein, we report an efficient, highly stable lubricant-infused coating for intranasal splints to render their surfaces antithrombogenic and repellent toward bacterial cells. Lubricant-infused intranasal splints were prepared by creating superhydrophobic polysiloxane nanofilament (PSnF) coatings using surface-initiated polymerization of n-propyltrichlorosilane (n-PTCS) and further infiltrating them with a silicone oil lubricant. Compared with commercially available intranasal splints, lubricant-infused, PSnF-coated splints significantly attenuated plasma and blood clot formation and prevented bacterial adhesion and biofilm formation for up to 7 days, the typical duration for which intranasal splints are kept. We further demonstrated that the performance of our engineered biointerface is independent of the underlying substrate and could be used to enhance the hemocompatibility and repellency properties of other medical implants such as medical-grade catheters.

Association of ABO blood group with bleeding severity in patients with bleeding of unknown cause

AbstractBlood group O has been associated with an increased bleeding tendency due to lower von Willebrand factor (VWF) and factor VIII (FVIII) levels. We explored whether blood group O is independently associated with bleeding severity in patients with mild-to-moderate bleeding of unknown cause (BUC) in the Vienna Bleeding Biobank cohort. Bleeding severity was recorded with the Vicenza bleeding score (BS). Blood group O was overrepresented in 422 patients with BUC compared with its presence in 23 145 healthy blood donors (47.2% vs 37.6%; odds ratio, 1.48; 95% confidence interval [CI], 1.22-1.79). The BS and the number of bleeding symptoms were significantly higher in patients with blood group O than in patients with non-O after adjustment for VWF and FVIII levels and sex (least-square [LS] means of BSs: 6.2; 95% CI, 5.8-6.6 vs 5.3; 4.9-5.7; and of number of symptoms: LS, 3.5; 95% CI, 3.2-3.7 vs 3.0; 2.8-3.2, respectively). Oral mucosal bleeding was more frequent in those with blood group O than in those with other blood types (group non-O; 26.1% vs 14.3%), independent of sex and VWF and FVIII levels, whereas other bleeding symptoms did not differ. Patients with blood group O had increased clot density in comparison with those with blood group non-O, as determined by rotational thromboelastometry and turbidimetric measurement of plasma clot formation. There were no differences in thrombin generation, clot lysis, or platelet function. Our data indicate that blood group O is a risk factor for increased bleeding and bleeding severity in patients with BUC, independent of VWF and FVIII levels.

Fibrin Clot Formation and Lysis in Plasma.

Disturbance in the balance between fibrin formation and fibrinolysis can lead to either bleeding or thrombosis; however, our current routine coagulation assays are not sensitive to altered fibrinolysis. The clot formation and lysis assay is a dynamic plasma-based analysis that assesses the patient’s capacity for fibrin formation and fibrinolysis by adding an activator of coagulation as well as fibrinolysis to plasma and measuring ex vivo fibrin clot formation and breakdown over time. This assay provides detailed information on the fibrinolytic activity but is currently used for research only, as the assay is prone to inter-laboratory variation and as it demands experienced laboratory technicians as well as specialized personnel to validate and interpret the results. Here, we describe a protocol for the clot formation and lysis assay used at our research laboratory.

Determination of fibrin clot growth and spatial thrombin propagation in the presence of different types of phospholipid surfaces

In this work, we present a new method—Thrombodynamics-4D—for the assessment of both plasma and platelet contributions to clotting. Thrombodynamics-4D potentially allows for the determination of plasma or platelet disorders and the effects of various drugs on plasma clotting or on platelet procoagulant function. In this assay, clot formation in platelet-rich plasma or platelet-free plasma supplemented with phospholipids is activated with tissue factor immobilized on a surface. Spatial fibrin clot growth and thrombin concentration dynamics are registered by measuring light scattering of the fibrin clot and fluorescence of the product formed by cleavage of the synthetic fluorogenic substrate by thrombin, respectively. Here, we describe the preanalytical requirements, measurement methodology and calculation principles of assay parameters. Preanalytical and analytical variability and reference ranges of the assay are given. Additionally, we show some clinical examples, which determine the effect of anticoagulants, measure clotting dysfunction in patients with platelet or coagulation disorders and evaluate the effect of surgery.

Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics.

Background: Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation.Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots.Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting.Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.