Rate Of Fibrin Polymerization Research Articles

Coagulation in Patients with Immune Thrombocytopenia and Other Associated Autoimmune Disorders

Introduction Chronic immune thrombocytopenia (ITP) is characterized by perturbations of immune homeostasis with hyperactivated effector cells as well as defective regulation of the adaptive immune system. It has been reported that, with varying frequency, patients with ITP may also have associated other autoimmune disorders, with circulating autoantibodies such as lupus anticoagulant antibodies (LACs), anti-nuclear antibodies (ANA), anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-beta2GPI), anti-thyroid peroxidase (ATPA), and rheumatoid factor (RF). Objectives To evaluate coagulation in patients with ITP with autoantibodies associated to other autoimmune diseases. Methods This is a prospective study that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. Adult ITP patients (n=182) without and with associated autoantibodies such as LACs, ANA, aCL, anti-beta2GPI, ATPA, and RF and healthy controls (n=180) were recruited. To evaluate the kinetics of clot formation and fibrinolysis, ROTEM® was performed on fresh platelet rich plasma adjusted to 25x10 9 platelets/L with the subjects own platelet poor plasma (PPP). Recalcification (NATEM) was used to induce coagulation. Clotting time (CT = time from start of measurement until initiation of clotting, in seconds); alpha angle (tangent to the curve at 2 mm amplitude, in degrees, which reflects the rate of fibrin polymerization); maximum clot firmness (MCF = maximum clot firmness, in mm, which reflects the maximum tensile strength of the thrombus); and lysis at 60 min (LI60) were recorded. Thrombin generation was measured in citrated PPP by calibrated automated thrombogram (CAT). To evaluate procoagulant capacity of plasma, coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/L of recombinant human tissue factor (TF) and 4 µmol/L of phospholipid mixture (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). Procoagulant activity of microparticles (MPs) associated with their content of either TF or phosphatidylserine (PS) was determined, respectively, with MP reagent (4 µM phospholipids) or PRP reagent (1 pM of recombinant human TF) by CAT. The following parameters were determined: Lag time (LT), or time from the start of the assay until 10 nM thrombin is formed, in min; time-to-peak (ttPeak), or time required to reach the maximum thrombin concentration, in min; peak height (Peak), or maximum thrombin concentration reached, in nM; and endogenous thrombin potential (ETP), or total amount of thrombin generated over time, in nMxmin. Results Fifty of the 182 patients with ITP (27.5%) were positive for autoantibodies associated with other autoimmune diseases, distributed as shown in Table 1. ANA were the most frequent antibodies present in these patients, and 22% had more than one kind of associated antibodies. Patients with ITP without antibodies had a shorter clot formation time (CT) than healthy controls. Presence of associated autoimmune diseases prolongs CT and resulting in values similar to controls. Both groups of patients with ITP had a diminished fibrinolysis (Table 2). Thrombin generation triggered by TF plus phospholipids as well as that dependent on TF content of microparticles (MP-reagent) was impaired in patients with other associated autoimmune disorders as shown by the prolonged LT and the diminution in either ETP or peak of generated thrombin (Table 2). When thrombin generation relayed on phospholipids of MPs, all patients with ITP generated less thrombin. Conclusion Association of other autoimmune diseases with ITP seemed to produce a delay in clot formation and in thrombin generation whereas did not affect the diminished fibrinolysis observed in ITP patients. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

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

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

Alteration of fibrin hydrogel gelation and degradation kinetics through addition of azo dyes

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength.

COVID-19 patient plasma demonstrates resistance to tPA-induced fibrinolysis as measured by thromboelastography.

Patients critically ill with COVID-19 are at risk for thrombotic events despite prophylactic anticoagulation. Impaired fibrinolysis has been proposed as an underlying mechanism. Our objective was to determine if fibrinolysis stimulated by tissue plasminogen activator (tPA) differed between COVID patients and controls. Plasma from 14 COVID patients on prophylactic heparin therapy was obtained and compared with heparinized plasma from 14 different healthy donors to act as controls. Kaolin activated thromboelastography with heparinase was utilized to obtain baseline measurements and then repeated with the addition of 4 nM tPA. Baseline fibrinogen levels were higher in COVID plasma as measured by maximum clot amplitude (43.6 ± 6.9 mm vs. 23.2 ± 5.5 mm, p < 0.0001) and Clauss assay (595 ± 135 mg/dL vs. 278 ± 44 mg/dL, p < 0.0001). With the addition of tPA, fibrinolysis at 30 min after MA (LY30%) was lower (37.9 ± 16.5% vs. 58.9 ± 18.3%, p = 0.0035) and time to 50% lysis was longer (48.8 ± 16.3 vs. 30.5 ± 15.4 min, p = 0.0053) in the COVID-19 samples. Clotting times and rate of fibrin polymerization (‘R’ or ‘α’ parameters) were largely the same in both groups. Clot from COVID patients contains a higher fibrin content compared to standard controls and shows resistance to fibrinolysis induced by tPA. These findings suggest the clinical efficacy of thrombolytics may be reduced in COVID-19 patients.

Citrullination of fibrinogen by peptidylarginine deiminase 2 impairs fibrin clot structure

BackgroundCitrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties. MethodsFibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen. ResultsCitrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots. ConclusionCitrullination of fibrinogen by PAD2 affects not only fibrin polymerization but also fibrin fiber properties, indicating that the fibrin network formed in the presence of cFBG may influence hemostasis. Our results suggest that citrullination of fibrinogen alters the composition of fibrin fibers which may lead to a looser fibrin network that is more susceptible to fibrinolysis and thereby affecting the hemostatic balance.

Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus.

Among complications of systemic lupus erythematosus (SLE), thrombotic events are relatively common and contribute significantly to the morbidity and mortality rates. An increased risk of thrombosis in various diseases has been shown to be associated with the lytic stability and mechanical stiffness of the fibrin clot determined by its structure. Here we studied alterations of the fibrin clot properties in relation to disease severity in SLE patients. Plasma clots from 28 SLE patients were characterized by the kinetics of formation and fibrinolytic dissolution (using dynamic turbidimetry), the network and fiber ultrastructure (scanning electron microscopy), viscoelasticity (shear rheometry), and the rate and degree of crosslinking (Western blotting) correlated with the disease activity, blood composition, and compared to clotting of pooled normal human plasma. Clots made from plasma of SLE patients were lysed faster with exogenous t-PA than control clots from normal plasma without a significant difference between those from active (SLEDAI>4) and inactive (SLEDAI<4) SLE patients. Clots from the blood of patients with active SLE were characterized by significantly slower onset, but faster rate of fibrin polymerization and a higher optical density due to thicker fibers compared to those from inactive SLE and control pooled normal plasma. The rheological parameters of the clots (storage and loss moduli) were significantly increased in the active SLE patients along with enhanced fibrin crosslinking and hyperfibrinogenemia. The structural and rheological alterations displayed a strong positive correlation with high fibrinogen levels and other laboratory markers of immune inflammation. In conclusion, changes in the blood composition associated with active systemic inflammation in SLE cause significant alterations in the lytic resistance of fibrin clots associated with changes in polymerization kinetics, viscoelastic properties, and structure. The formation of more rigid prothrombotic fibrin clots in the plasma of SLE patients is likely due to the inflammatory hyperfibrinogenemia and greater extent of crosslinking. However, the higher susceptibility of the SLE clots to fibrinolysis may be a protective and/or compensatory mechanism that reduces the risk of thrombotic complications and improves patient outcomes.

Comparison of antithrombin‐dependent and direct inhibitors of factor Xa or thrombin on the kinetics and qualitative characteristics of blood clots

BackgroundVenous thromboembolism (VTE) is associated with significant morbidity and mortality. ObjectivesWe investigated the impact of direct and AT‐dependent FXa or thrombin inhibitors on thrombus formation. MethodsWhole blood thromboelastometry and thrombin generation were assessed after triggering the TF pathway. Clinically relevant concentrations of rivaroxaban, fondaparinux, dabigatran or tinzaparin and an association of rivaroxaban and dabigatran were examined. ResultsAll agents delayed thrombus formation in a concentration‐dependent manner, as documented by the prolongation of the clotting time (CT) and clot formation time (CFT). Rivaroxaban did not significantly alter the α‐angle or maximum clot firmness (MCF). In contrast, dabigatran and fondaparinux altered the process of clot structure by decreasing the α‐angle, but did not modify clot firmness. The later property was significantly affected only by tinzaparin that also reduced the MCF. The association of rivaroxaban and dabigatran did not affect the MCF, although it amplified the effect on CFT and α‐angle. ConclusionsAll agents delayed thrombus formation. However, the compounds differed substantially with respect to fibrin polymerization rate and clot firmness. Comparison of the data obtained by thrombin generation assessment with those obtained by the thromboelastometric study shows that the delay in clot formation is principally associated with prolongation of the initiation phase of thrombin formation as well as a reduction of the propagation phase. Tinzaparin was much more potent than the other agents both with regard to suppression of thrombin generation and by delay in clot formation.

Haemostasis in Immune Trombocytopenia Patients Responders to Agonists of Thrombopoietin Receptor

Introduction: The goal of treatment of patients with immune thrombocytopaenia (ITP) is to raise platelet count to a level that will minimize or stop bleeding. One of the therapeutic strategies employed is to augment platelet production with agonists of the thrombopoietin receptor (TPOR-A). Clinical trials for TPOR-A comparing their effects with placebo showed a reduction in proportion of patients reporting bleeding (Tarantino et al, Blood Coag Fibrinolysis, 2013, 24:284-296). This effect might rely on the increase in platelet count, but other factors known to compensate bleeding risk in thrombocytopenic ITP patients might be regulated by TPOR-A.Objective: We aimed to compare haemostasis in ITP patients untreated with any pharmacologic agent (UT-ITPp) and treated with TPOR-A (TPOA-Rp) using the coagulation global assays thromboelastrometry (ROTEM®) and Calibrated Automated Thrombogram (CAT).Methods: Thirty chronic UT-ITPp [platelet count: (86±60)x109 platelets/L], twenty six responders to TPOR-A [16 with Romiplostim® and 10 with Revolade®, platelet count: (132±46)x109 platelets/L] and fifty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). Aliquots were stored at -80ºC until analysis.Non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.Plasma thrombin generation was measured in PPP using the CAT test at a final concentration of 1 pM tissue factor and 4 microM phospholipids. We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak.Platelet activation was determined by flow cytometry through PAC1-FITC binding after stimulation with 100 micromol/L thrombin receptor-activating peptide 6 (TRAP). Fibrinolytic proteins were tested in PPP using commercialized kits.Comparisons of quantitative variables were made with SPSS.22 software. Values of p≤0.05 were considered statistically significant.Results: Platelets from all patients with ITP had a defect in their ability to be activated, as shown by the lower PAC1 binding (p<0.001).No differences were observed among groups for thrombin generation except for an increase in ETP in both groups of ITP patients [control: 1240±320 nMxmin, UT-ITPp: 1464±443 nMxmin (p<0.05), TPOA-Rp: 1533±390 nMxmin (p<0.05)].In ROTEM experiments, PRP from UT-ITPp and TPOA-Rp showed a prolonged CT [control: 550± 95 sec, UT-ITPp: 1090±75 sec (p<0.05), TPOA-Rp: 859±209 sec (p<0.05)]. Only UT-ITPp presented a diminished alpha angle [control: 60.3±7.5, UT-ITPp: 49.3±7.3 (p<0.05), TPOA-Rp: 55.8±5.58]. On the other hand, TPOA-Rp had an increased MCF (control: 46.5±2.1 mm, UT-ITPp: 50.2±5.6 mm, TPOA-Rp: 57.9±4.4 mm, p<0.05) and LI60 (control: 91.8±3.1 %, UT-ITPp: 94.1±3.1 %, TPOA-Rp: 97.6±1.1 %, p<0.05).To evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed among the two groups of patients and healthy controls except for PAI-1 which level was increased in TPOA-Rp (control: 12.9±12.3 ng/ml, UT-ITPp: 17.3±12.5 ng/ml, TPOA-Rp: 46.8±20.8, ng/ml p<0.05).Conclusions: As we previously described, TPOR-A treatment increased platelet count but did not ameliorate their function (Álvarez Román et al, Thromb Haemost. 2014, 112:65-72). Nevertheless, ITP patients responders to TPOR-A showed a haemostasis unbalanced on a hypercoagulable profile due, at least in part, to a hipofibrinolytic pattern mainly caused by an increase in PAI-1 plasma level. This fact, together with the increment in platelet count caused by TPOR-A, might help to protect these patients from bleeding.This work was supported by a grant from the FIS-FEDER PI15/01457 DisclosuresNo relevant conflicts of interest to declare.

Procoagulant Profile in Patients with Immune Thrombocytopaenia

Background: Patients with platelet counts less than 20 or 30 x 109/L have an increased risk of bleeding. Nevertheless, some patients with immune thrombocytopenia (ITP) have fewer bleeding symptoms than expected. In a previous communication (ASH 2014) we reported that these patients presented high microparticles (MP)-associated procoagulant activity to compensate bleeding risk and that cellular origin of these MPs were platelets and red cells. However, other mechanisms might be involved.Objective: The aim of this work was to analyse the involvement of other factors to compensate bleeding risk in thrombocytopenic ITP patients. Moreover, the feasibility of using the coagulation global assays thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT) to test haemostasis in these patients was evaluated.Methods: Fifty patients with chronic ITP with platelet count less than 50 x 109/L and twenty-five healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP) and aliquots were stored at -70ºC until analysis.To assess the kinetics of clot formation, non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness,which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.Plasma thrombin generation was measured in PPP using the Calibrated Automated Thrombogram (CAT) test at a final concentration of 1 pM tissue factor and 4 mM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak.Fibrinolytic proteins and E-selectin was tested in PPP using commercialized kits.Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant.Results: PRP from ITP patients showed a prolonged CT (control: 550+ 95 sec, ITP: 890+165 sec, p<0.01), diminished alpha angle (control: 62.8+4.3, ITP: 53.5+7.5, p<0.05), and increased MCF (control: 46.7+3.1mm, ITP: 52.4+6.1 mm, p<0.05) and LI60 (control: 90.6+3.0% , ITP: 95.5+3.4, p<0.05) when compared with controls.In order to evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed between patients and healthy controls except for PAI-1 which level was increased in ITP patients (control: 14.7 ng/ml+11.7 ng/ml, ITP: 30.4+17.5, p<0.05). Since plasma PAI-1 might be increased as consequence of endothelial damage, plasma concentration of E-selectin, marker of endothelial injury, was determined. E-selectin was increased in samples from ITP patients (control: 10.5 ng/ml+3.9 ng/ml, ITP: 31.6+14.0, p<0.05). Moreover, MCF and LI60 ROTEM parameters correlated to E-selectin plasma concentration (Spearman r values 0.6643, p<0.001 for MCF; 0.6053, p<0.001 for LI60).Thrombin generation in PPP was also measured and a shorter time to peak (control: 9.3+1.2 sec, ITP: 8.3+1.7 sec,p<0.05) and increased ETP (control: 1223.8+257.7 nMxmin, ITP: 1696.4+524 nMxmin,p<0.05) and peak (control: 225.7+82.8.1 nM, ITP: 330.4+106.1 nM,p<0.05) were observed in ITP patients.Conclusions: We demonstrated that ITP patients presented a hypercoagulable profile that might be related, at least in part, to a reduced fibrinolysis mainly caused by an increase in PAI-1 level that seemed to be related to endothelial damage. Moreover ROTEM and CAT appeared to be useful tools for evaluating coagulant profile in ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Haemostasis Impairment in Patients with Myelodysplastic Syndromes with Normal Platelet Counts

Background: Bleeding complications can have serious outcomes in patients with myelodysplastic syndromes (MDS) and are usually related to thrombocytopaenia. In a recent work we demonstrated that platelets from MDS have impaired capacity to respond to agonist stimulation and exposed more phosphatidylserine (PS) than those from healthy controls whichever their platelet count was (Martin et al, Thromb Haemost., 2013;109:909-19). The exposure of PS in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes and thrombin generation. So, the possibility exists that these processes are increased in these patients.Objectives: The aim of this work was to study whether thrombus formation and thrombin generation is impaired in MDS patients with normal platelet count employing global coagulation tests thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT).Methods: Thirty-one MDS patients with normal platelet count and twenty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). For ROTEM and CAT experiments, PRP was adjusted to a platelet count of 25 x 109/L. Aliquots for ROTEM assay were tested within the two hours after obtaining blood samples. For CAT experiments, adjusted PRP and PPP aliquots were stored at -70ºC until analysis.Kinetics of clot formation, non-activated ROTEM was performed on adjusted PRP. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), and maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), were recorded.Thrombin generation was measured in adjusted PRP without any trigger and in PPP with 1 pM tissue factor and 4 µM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands) as trigger. Endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak were evaluated.Platelet activation was determined by PAC1 (BD, Madrid, Spain) binding after stimulation with 100 μM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) and surface PS through Annexin-V binding and flow cytometry analyses.Results: Platelets from MDS patients had a reduced response to TRAP stimulation (control= 12016+6384 arbitrary units; MDS= 5829+3704 arbitrary units) and exposed more PS than controls (control= 362.1+80.5 MF; MDS= 378.5+173.5 MF).ROTEM experiments showed kinetic parameters corresponding to a hipocoagulable profile (CT: control= 550+95 sec, MDS= 922+216 sec, p<0.01; alpha angle control= 62.8+4.3, MDS= 47.5+6.0, p<0.05; A5: control= 29.8+3.2, MDS= 24.2+5.8, p<0.05 whereas MCF was similar to controls (control= 46.7+3.1mm, MDS= 47.5+4.3 mm).In order to evaluate whether the impaired clot formation was due to a reduction in plasma-associated thrombin generation, CAT experiments were performed in PPP samples. No differences were found between MDS patients and control group (ETP: control=1223.4+257.8 nMxmin, MDS= 1224.4+344 .1 nMxmin; peak: control= 279.5+54.7nM, MDS= 265.9+64.1 nM). On the other hand, when thrombin generation experiments were performed in adjusted PRP, ETP and peak values were lower than in control group (ETP: control=1574.8+430.5 nMxmin, MDS= 1167.4+354 nMxmin,p<0.05; peak: control= 225.6+82.9 nM, MDS= 266.9+92.2 nM, p<0.05).Conclusions: Platelet dysfunction might be resposible of bleeding complications observed in patients with MDS with normal platelet counts. Increment in PS exposure on platelet surface did not seem to compensate impairment in platelet function. DisclosuresNo relevant conflicts of interest to declare.

Thromboelastometry Shows a Prothrombotic State in Systemic Lupus Erythematosus Related to Diminished Fibrinolysis and Microparticle-Derived Tissue Factor

Background: Systemic Lupus Erythematosus (SLE) is an autoimmune disorder of unknown origin with a high mortality pattern due to the development of a premature cardiovascular disease. The presence in these patients of a dysfunctional endothelium together with a hypercoagulable milieu may contribute to an increased incidence of thrombotic events. Increased thrombin generation, elevated levels of circulating microparticles and plasmatic levels of PAI-1 may contribute to the prothrombotic phenotype of the disease but data are scarce.Objectives: 1-To characterize the prothrombotic state in SLE by rotational thromboelastometry (ROTEM) and thrombin generation linked to tissue factor bearing microparticles. 2- To evaluate endothelial damage in patients with SLE and its relationship with the prothrombotic state of the disease. 3- To evaluate the influence of PAI-1 in the prothrombotic state of SLE.Material and methods: 39 patients with SLE and 25 sex and age matched healthy subjects were included. Whole blood was drawn in standard BD sodium citrate tubes (3.2%). ROTEM was performed in naTEM condition. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), the time that clot takes to increase from 2mm above baseline to 20mm above baseline (CFT) and A5, amplitude at 5 min, were recorded.To evaluate the presence of tissue factor bearing microparticles, thrombin generation was determined by Calibrated Automated Thrombogram (CAT) in presence of 4 mM phospholipid (MP-Reagent, Diagnostica Stago, Spain). The endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak were evaluated.Antigenic levels of E-selectin and PAI-1 were determined by ELISA (R&D Systems, MN, USA and Affymetrix eBioscience, Vienna, Austria) respectively.Results: ROTEM parameters showed a hypercoagulable profile in LES patients. CT and CFT were shorter (p<0.001 in both cases), and MCF, alpha angle and A5 were higher (p<0.001, p<0.02 and p<0.001 respectively) when compared to healthy controls. On the other hand, CAT parameters did not show differences between both groups. Nevertheless, in LES patients but not in controls, CAT parameters significantly correlated with ROTEM ones (Table 1). Table 1Correlations in the LES patients group between CAT and ROTEM parameters. Analyses were performed with Spearman test. N: number of determinations; p* denotes significance.CTCFTMCFMCF-talphaA5A10ETP(MP)r-,301-,375*,369*-,368*,378*,364*,393*p,0840,028*0,031*0,032*0,027*0,034*0,021*N34343434343434Peak(MP)r-,353*-,396*,372*-,345*,396*,393*,402*p0,040*0,020*0,030*0,045*0,020*0,021*0,018*N34343434343434ttPeak(MP)r,240,222-,242,217-,223-,209-,210p,171,207,168,217,205,236,232N34343434343434In order to evaluate endothelial damage, plasma E-selectin and PAI-1 were determined. No differences were found in E-selectin level whereas increased PAI-1 levels were seen in LES group (p<0.006). PAI-1 did not correlate to ROTEM parameters.Conclusions: ROTEM can detect a hypercoagulable state in patients with SLE. The hypercoagulable state may be linked to increased tissue factor bearing microparticles and increased PAI-1 plasma levels. DisclosuresNo relevant conflicts of interest to declare.

Effect of von Willebrand factor on clot structure and lysis.

Von Willebrand Factor (vWF) is constitutively secreted by the endothelium and incorporated in the fibrin clots under slow clotting conditions. The aim of the present work was to study the effect of vWF on clot structure and lysis. Purified fibrinogen was mixed with vWF or Tris-buffered saline and clotted with thrombin - activated factor XIII. Fibrin polymerization was followed by turbidity at 350 nm during 2.5 h. After this time, plasmin was added on the top of the clots, and the optical density (OD) was read until baseline values. vWF effect on network[Combining Acute Accent]s porosity was evaluated by permeation using the same clotting conditions as for fibrin polymerization. Clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM). The rate of fibrin polymerization was 1.47 mOD/s in the presence of vWF and 0.5 mOD/s when vWF was not added (P < 0.05). The fibrin lysis rate was approximately four times faster when vWF was added to fibrinogen. The fibrin network porosity was (20.4 ± 1.6) × 10 cm with vWF and (8.3 ± 1.2) × 10 cm without external vWF (P < 0.05). The analysis of LSCM images showed that vWF increased fibrin fibers diameter and the networks[Combining Acute Accent] pores size. In conclusion, vWF covalently crosslinked to fibrin modify its structure (increases fibrin diameter and the pores filling space of the meshwork) that accelerates the fibrin lysis rate.

Variation of fibrinogen oligosaccharide structure in the acute phase response: Possible haemorrhagic implications

BackgroundFibrinogen is an acute phase glycoprotein whose concentration increases in response to trauma. The newly synthesised protein is functionally enhanced and it is known that treatment with neuraminidase increases the rate of fibrin polymerisation. To explore this, we examined the differences between the oligosaccharide structures of quiescent and acute phase fibrinogen. MethodsA series of plasma samples was obtained from two individuals suffering an acute phase response. Fibrinogen chains were examined directly by ESI mass spectrometry before and after digestion with N-glycosidase F and β1,4 galactosidase. ResultsThe Bβ and γ chains of acute phase fibrinogen showed a mass decrease of 162Da (Gal) in some 50% of the molecules, and the Bβ chain showed an additional decrease corresponding to a further loss of NAcGlc. Incubation with N-glycosidase F normalised all isoform masses to that of the quiescent naked protein, confirming the N-linked oligosaccharide as the source of heterogeneity. β1,4 galactosidase treatment showed the structural difference was the absence of the penultimate Gal from the biantennary oligosaccharides, and mapping of tryptic glycopeptides confirmed these results showing that approximately half the chains lacked Gal. Conclusions and implicationsThe failure of incorporation Gal excludes the possibility of the hepatic NAcNeu Gal transferase capping the oligosaccharides with sialic acid. This has two desirable haemostatic outcomes: fibrin monomers will polymerise and form clots more rapidly, and two galactose residues can never be exposed diminishing uptake of the protein by the asialoglycoprotein receptor and ramping up concentration at a time of challenge.

Comparative studies of purified poloxamer 188 using clot‐based and viscoelastic measurements of coagulation (833.5)

Poloxamer 188 (P188; Mast Therapeutics, Inc.)is a surface‐active, non‐ionic block copolymer which binds to hydrophobic surfaces on damaged cells improving membrane hydration and lowering adhesion and viscosity. We compared the effect of this agent using various clot based, chromogenic and viscoelastic measurements of blood coagulation. Whole blood activated clotting time ACT) studies were carried out in healthy individuals (n=10) at a concentration range of 1.872‐15.0mg/mL. TEG analysis was carried out on TEG 5000 (Haemoscope Corp, Niles, IL) at concentrations of 0‐0.45 mg/mL. The effect of P188 on normal plasma clotting parameters, such as PT and aPTT, was measured at a concentration range of 0‐10 mg/mL. The effect of P188 on thrombin‐induced clot formation was investigated using a fibrinokinetic method. The effect of P188 on thrombin generation was measured using the fluormetric method (Technoclone, Vienna, Austria). The anti‐protease effects of P188 were studied using chromogenic substrate methods using isolated biochemical systems. At concentrations up to 10 mg/mL, P188 did not produce any modification of ACT or the standard clotting assays. In the TEG analysis, P188 produced a concentration dependent hypocoagulant effect in as evidenced by increased angle and shortening of maximum amplitude (MA). In fibrinokinetic studies, P188 produced an increase in the fibrin clot density and rate of fibrin polymerization. The marked discordance between TEG and other coagulation tests suggest that P188’s effect on viscosity and adhesive interactions result in an artifact in TEG analysis and an incorrect indication of a hypocoagulant effect.

Calixarene methylene bisphosphonic acids as promising effectors of biochemical processes

this interdisciplinary study, performed with participation of research workers of Palladin Institute of Biochemistry and Institute of organic chemist ry of NaS of ukraine, is devoted to analysis of biochemical effects of some calixarene methylene bisphosphonic acids (cyclic phenol oligomers) on two well-known biological phenomenons – mg2+-dependent atP hydrolysis (myosin subfragment-1 of myometrium smooth muscle was used as an example) and fibrin polymerization. calix[4]arene С-97 (calix[4]arene methylene bisphosphonic acids) is a macrocyclic substance, which contains intramolecular highly ordered lipophilic cavity formed by four aromatic rings, one of which is functionalized at the upper rim with methylene bisphosphonic group. at concentration of 100 μm, this substance was shown to effectively inhibit atPase activity of pig myometrium myosin subfragment-1 (inhibition coefficient І0.5 = 83 ± 7 μm). at the same time, this calix[4]arene causes significant (vs. control) increase of myosin subfragment-1 hydrodynamic diameter, which may indicate formation of an intermolecular complex between calixarene and myosin head. computer simulation methods (docking and molecular dynamics with addition of grid technologies) enabled to elucidate the grounds of intermolecular interactions between calix[4]arene С-97 and myometrium myosin subfragment-1, that involve hydrophobic, electrostatic and π-π-stacking interactions, some of which are close to the atPase active centre. In view of the ability of calixarenes to penetrate into the cell and their low toxicity, the results obtained may be used as a basis for further development of a new generation of supramolecular effectors (starting from the above mentioned substances, in particular calix[4]arene С-97) for regulation of smooth muscle contractile activity at the level of atP dependent actin-myosin interaction. calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. the most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (c-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52·10-6 m (Ic50 ). at this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desaB fibrin polymerization, the Ic50 was 1.26·10 -6 m at a molar ratio of c-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]-arene bis-methylene-bis-phosphonic acid (c-98) inhibited fibrin desaB polymerization with an Ic50 = 1.31·10 -4 m. We hypothesized that c-192 blocks fibrin formation by combining with polymerization site ‘a’ (aa17–19), which ordinarily initiates protofibril formation in a ‘knob-hole’ manner. this suggestion was confirmed by an hPLc assay, which showed a host–guest inclusion complex of c-192 with the synthetic peptide gly-Pro-arg-Pro, an analogue of site ‘a’. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. calixarene c-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13·10-5 and 1.10·10-5 m, respectively. these experiments demonstrate that c-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.

Comparative Studies Of Purified Poloxamer 188 Using ClotBased and Viscoelastic Measurements Of Coagulation

Background Poloxamer 188 (P188; Mast Therapeutics, Inc.) is a surface-active, non-ionic block copolymer which binds to hydrophobic surfaces on damaged cells improving membrane hydration and lowering adhesion and viscosity. It is known to improve microvascular function in various pathologic states. Currently, this agent is under investigation in a phase 3 clinical trial for treatment of sickle cell disease patients experiencing acute vaso-occlusive crisis. The effect of P188 on blood coagulation and platelet function has been evaluated in several clinical trials where no clinically significant effect has been observed. In these trials, coagulation studies were based on standard clot based methods (e.g., PT and aPTT) and did not include viscoelastic measurements such as thromboelastography (TEG). Given P188 alters viscosity, we compared the effect of this agent using various clotbased, chromogenic and viscoelastic measurements of blood coagulation. Materials and Methods Whole blood activated clotting time studies were carried out in groups of healthy individuals (n=10) at a concentration range of 1.872-15.0mg/mL. TEG analysis on native and citratedwhole blood was carried out on TEG 5000 (Haemoscope Corp, Niles, IL) at concentrations of 0-0.45 mg/mL. The effect of P188 on normal plasma clotting parameters, such as PT and aPTT, was measured at a concentration range of 0-10 mg/mL. The effect of P188 on thrombin-induced clot formation was investigated using a fibrinokinetic method. The effect of P188 on thrombin generation was measured using the fluormetric method (Technoclone, Vienna, Austria). The anti-protease effects of P188 were studied using chromogenic substrate methods using isolated biochemical systems. Results At concentrations up to 10 mg/mL, P188 did not produce any modification of Celiteactivated clotting time (Celite-ACT). At all concentrations the Celite-ACT values remained comparable to saline (138-140 sec for P188 vs. 140 sec for saline). In the TEG analysis, P188 produced a concentrationdependent hypocoagulant effect in both native and citratedblood as evidenced by increased angle and shortening of maximum amplitude (MA). In standard PT and aPTT tests, P188 did not produce any effect on the clotting profile at concentrations up to 20 mg/mL. In fibrinokinetic studies, P188 produced an increase in the fibrin clot density and rate of fibrin polymerization. Discussion These studies demonstrate that even at very high concentrations, P188 does not produce an effect on whole blood clotting as measured by the Celite-ACT assay; this result was confirmed in other standard assays. Fibrinokinetic studies revealed an increase in the rate of fibrin formation and clot density. However, at relatively low concentrations, P188 exhibited a hypocoagulant profile in TEG analysis. The marked discordance between TEG and other coagulation tests suggest that P188’s effect on viscosity and adhesive interactions result in an artifact in TEG analysis and an incorrect indication of a hypocoagulant effect. This effect may be due to the viscoelastic endpoint in the TEG assay. Further studies are needed to confirm this hypothesis. Disclosures: Fareed: Mast Therapeutics: Research Funding. Emanuele:Mast Therapeutics: Employment.

Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization

Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10(-6) M (IC(50)). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC(50) was 1.26 × 10(-6) M at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC(50) = 1.31 × 10(-4) M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site 'A' (Aα17-19), which ordinarily initiates protofibril formation in a 'knob-hole' manner. This suggestion was confirmed by an HPLC assay, which showed a host-guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site 'A'. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10(-5) M and 1.10 × 10(-5) M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.

The specific interaction between fibrin(ogen) and hyaluronan: possible consequences in haemostasis, inflammation and wound healing.

We have proposed that fibrin and hyaluronan (HA) are macromolecular regulators during inflammation and wound healing. Here we extend our studies to characterize the specific interaction between fibrin(ogen) and HA. 125I-labelled HA (Mr approximately 32,000) was bound by plastic surfaces coated with human fibrinogen but not bovine serum albumin, ovalbumin, beta-lactoglobin or rabbit immunoglobulin G. 125I-labelled fibrinogen bound to a unique hexylamine derivative of HA coupled to Sepharose and was eluted specifically by HA oligosaccharides in a size-dependent manner. A dot blot assay, in which proteins are adsorbed to nitrocellulose and probed with 125I-HA, also showed specific binding to human fibrinogen. This assay was used to examine fibrinogens from other mammalian species. No specific 125I-HA binding was observed with the protein from horse, rat or cow. Significant binding was detected with human, sheep, rabbit, dog, baboon, goat and pig fibrinogens. Thrombin-induced formation of fibrin clots is also affected by HA, which decreases the lag time before clotting and increases the rate of clot formation. The rate of fibrin polymerization increased over 500% in the presence of 60 microM HA. Furthermore, the structure of the fibrin gel, as assessed by light scattering, was altered by HA or chondroitin sulphate in a concentration-dependent manner. The results support the proposed wound-healing model and indicate that an increase in circulating HA levels could adversely affect haemostasis and increase the risk of thrombosis or bleeding. The interaction between HA and fibrinogen emphasizes the importance of the liver endothelial cell HA receptor in the removal of glycosaminoglycans from the blood. Cultured cells continuously endocytosing 125I-HA for 4 h reutilized their total cellular HA receptors at least once every 50 min even in the presence of cycloheximide. This endocytotic receptor was therefore shown to be part of a recycling system.

The ultrastructure of fibrinogen-420 and the fibrin-420 clot

Fibrinogen-420 is a minor subclass of human fibrinogen that is so named because of its higher molecular weight compared to fibrinogen-340, the predominant form of circulating fibrinogen. Each of the two Aα chains of fibrinogen-340 is replaced in fibrinogen-420 by an Aα isoform termed α E. Such chains contain a globular C-terminal extension, α EC, that is homologous with the C-terminal regions of Bβ and γ chains in the fibrin D domain. The α EC domain lacks a functional fibrin polymerization pocket like those found in the D domain, but it does contain a binding site for β 2 integrins. Electron microscopy of fibrinogen-340 molecules showed the major core fibrinogen domains, D–E–D, plus globular portions of the C-terminal αC domains. Fibrinogen-420 molecules had two additional globular domains that were attributable to α EC. Turbidity measurements of thrombin-cleaved fibrinogen-420 revealed a reduced rate of fibrin polymerization and a lower maximum turbidity. Thromboelastographic measurements also showed a reduced rate of fibrin-420 polymerization (amplitude development) compared with fibrin-340. Nevertheless, the final amplitude (MA) and the calculated elastic modulus ( G) for fibrin-420 were greater than those for fibrin-340. These results suggested a greater degree of fibrin-420 branching and thinner matrix fibers, and such structures were found in SEM images. In addition, fibrin-420 fibers were irregular and often showed nodular structures protruding from the fiber surface. These nodularities represented α EC domains, and possibly αC domains as well. TEM images of negatively shadowed fibrin-420 networks showed irregular fiber borders, but the fibers possessed the same 22.5-nm periodicity that characterizes all fibrin fibers. From this result, we conclude that fibrin-420 fiber assembly occurs through the same D–E interactions that drive the assembly of all fibrin fibrils, and therefore that the staggered overlapping molecular packing arrangement is the same in both types of fibrin. The α EC domains are arrayed on fiber surfaces, and in this location, they would very likely slow lateral fibril association, causing thinner, more branched fibers to form. However, their location on the fiber surface would facilitate cellular interactions through the integrin receptor binding site.

Citrated Blood Does Not Reliably Reflect Fresh Whole Blood Coagulability in Trials of In Vitro Hemodilution

Citrated Blood Does Not Reliably Reflect Fresh Whole Blood Coagulability in Trials of In Vitro Hemodilution