Contact Phase Activation Research Articles

Bioactive surface coating for preventing mechanical heart valve thrombosis

BackgroundProsthetic heart valves are the only treatment for most patients with severe valvular heart disease. Mechanical valves, made of metallic components, are the most long-lasting type of replacement valves. However, they are prone to thrombosis and require permanent anticoagulation and monitoring, which leads to higher risk of bleeding and impacts the patient’s quality of life. ObjectivesTo develop a bioactive coating for mechanical valves with the aim to prevent thrombosis and improve patient outcomes. MethodsWe used a catechol-based approach to produce a drug-releasing multilayer coating adherent to mechanical valves. The hemodynamic performance of coated Open Pivot valves was verified in a heart model tester, and coating durability in the long term was assessed in a durability tester producing accelerated cardiac cycles. Coating antithrombotic activity was evaluated in vitro with human plasma or whole blood under static and flow conditions and in vivo after surgical valve implantation in a pig’s thoracic aorta. ResultsWe developed an antithrombotic coating consisting of ticagrelor- and minocycline-releasing cross-linked nanogels covalently linked to polyethylene glycol. We demonstrated the hydrodynamic performance, durability, and hemocompatibility of coated valves. The coating did not increase the contact phase activation of coagulation, and it prevented plasma protein adsorption, platelet adhesion, and thrombus formation. Implantation of coated valves in nonanticoagulated pigs for 1 month efficiently reduced valve thrombosis compared with noncoated valves. ConclusionOur coating efficiently inhibited mechanical valve thrombosis, which might solve the issues of anticoagulant use in patients and the number of revision surgeries due to valve thrombosis despite anticoagulation.

The Characteristics of Dialysis Membranes: Benefits of the AN69 Membrane in Hemodialysis Patients.

Patients undergoing hemodialysis (HD) experience serious cardiovascular complications, through malnutrition, inflammation, and atherosclerosis. Amputation for peripheral arterial disease (PAD) is more prevalent in patients undergoing HD than in the general population. In addition, revascularization procedures in dialysis patients are often associated with subsequent amputation and high mortality rates. To improve the prognosis of dialysis patients, malnutrition and inflammation must be properly treated, which necessitates a better understanding of the characteristics of dialysis membranes. Herein, the characteristics of several dialysis membranes were studied, with a special reference to the AN69 membrane, noting several similarities to low-density lipoprotein (LDL)-apheresis, which is also applicable for the treatment of PAD. Both systems (LDL-apheresis and AN69) have anti-inflammatory and anti-thrombogenic effects because they use a negatively charged surface for extracorporeal adsorptive filtration from the blood/plasma, and contact phase activation. The concomitant use of both these therapeutic systems may have additive therapeutic benefits in HD patients. Here, we reviewed the characteristics of dialysis membranes and benefits of the AN69 membrane in dialysis patients.

Hemostasis in neonatal ECMO.

Extracorporeal membrane oxygenation (ECMO) is a life-saving support for cardio-respiratory function. Over the last 50 years, the extracorporeal field has faced huge technological progress. However, despite the improvements in technique and materials, coagulation problems are still the main contributor to morbidity and mortality of ECMO patients. Indeed, the incidence and survival rates of the main hemorrhagic and thrombotic complications in neonatal respiratory ECMO are relevant. The main culprit is related to the intrinsic nature of ECMO: the contact phase activation. The exposure of the human blood to the non-endothelial surface triggers a systemic inflammatory response syndrome, which chronically activates the thrombin generation and ultimately leads to coagulative derangements. Pre-existing illness-related hemostatic dysfunction and the peculiarity of the neonatal clotting balance further complicate the picture. Systemic anticoagulation is the management's mainstay, aiming to prevent thrombosis within the circuit and bleeding complications in the patient. Although other agents (i.e., direct thrombin inhibitors) have been recently introduced, unfractionated heparin (UFH) is the standard of care worldwide. Currently, there are multiple tests exploring ECMO-induced coagulopathy. A combination of the parameters mentioned above and the evaluation of the patient's underlying clinical context should be used to provide a goal-directed antithrombotic strategy. However, the ideal algorithm for monitoring anticoagulation is currently unknown, resulting in a large inter-institutional diagnostic variability. In this review, we face the features of the available monitoring tests and approaches, mainly focusing on the role of point-of-care (POC) viscoelastic assays in neonatal ECMO. Current gaps in knowledge and areas that warrant further study will also be addressed.

Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

IntroductionHematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment.We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. MethodsThis is a single-centre, case-control, non-interventionist, prospective study.During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed:Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway.Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software.TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA).The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. ResultsThose patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests.Table 1 shows the results obtained.Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI.Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI.Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. ConclusionsUBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient.Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes.TFPI inhibition might emerge as a good therapy for some of these patients.Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field.This work was supported by Novo Nordisk Pharma S.A.Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LTlagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. [Display omitted] DisclosuresAlvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

A Novel C1-Esterase Inhibitor Oxygenator Coating Prevents FXII Activation in Human Blood.

The limited hemocompatibility of currently used oxygenator membranes prevents long-term use of artificial lungs in patients with lung failure. To improve hemocompatibility, we developed a novel covalent C1-esterase inhibitor (C1-INH) coating. Besides complement inhibition, C1-INH also prevents FXII activation, a very early event of contact phase activation at the crossroads of coagulation and inflammation. Covalently coated heparin, as the current anticoagulation gold standard, served as control. Additionally, a combination of both coatings (C1-INH/heparin) was established. The coatings were tested for their hemocompatibility by dynamic incubation with freshly drawn human whole blood. The analysis of various blood and plasma parameters revealed that C1-INH-containing coatings were able to markedly reduce FXIIa activity compared to heparin coating. Combined C1-INH/heparin coatings yielded similarly low levels of thrombin-antithrombin III complex formation as heparin coating. In particular, adhesion of monocytes and platelets as well as the diminished formation of fibrin networks were observed for combined coatings. We could show for the first time that a covalent coating with complement inhibitor C1-INH was able to ameliorate hemocompatibility. Thus, the early inhibition of the coagulation cascade is likely to have far-reaching consequences for the other cross-reacting plasma protein pathways.

Laboratory investigation of a rare coagulation factor deficiency

We present a case report of a 30-year-old pregnant patient who presented for haematologist review following incidental finding of a prolonged APTT. There was no family history of bleeding or thrombosis. Lupus anticoagulant testing, mixing study and factor assays were requested. Laboratory testing revealed a deficiency of prekallikrein. Prekallikrein is a serine protease which forms part of the contact activation phase of coagulation. Prekallikrein deficiency is an autosomal recessive trait, and patients with homozygous or compound heterozygous deficiency will present with plasma prekallikrein levels <1%. While it is understood that patients with prekallikrein deficiency do not have a bleeding tendency, the defect may have a role in the development of hypertension and other cardiovascular conditions. This case study will review the laboratory investigations necessary to diagnose prekallikrein deficiency, and the sensitivity of different APTT reagents to deficiency of prekallikrein.

Inflammation and coagulation following minimally invasive extracorporeal circulation technologies.

Minimally invasive extracorporeal perfusion technologies are based on the use of a minimally invasive extracorporeal circulation (MiECC) system. This includes a closed CPB circuit; biologically inert blood contact surfaces; reduced priming volume; a centrifugal pump; a membrane oxygenator; a heat exchanger; a cardioplegia system; a venous bubble trap/venous air removing device; and a shed blood management system. Some of these items, alone or in combination, are able to modify the blood activation usually elicited by cardiopulmonary bypass (CPB). The hemostatic system activation is less activated and lower degrees of thrombin generation and platelet activation have been found in numerous studies. Additionally, the reduced level of hemodilution plays an important role in preserving clot firmness after CPB with MiECC. These biochemical changes are reflected by a blood loss containment, a reduced need for allogeneic blood transfusions, and, in some studies, by a lower thromboembolic complications rate. The activation of the inflammatory cascade is in turn limited by MiECC, both directly (through a blunting of the contact-phase activation) and indirectly (through a limited thrombin generation, platelet activation, and consequent lower release of pro-inflammatory cytokines). The clinical consequences of this are mainly demonstrated by a lower rate of postoperative atrial fibrillation; other inflammation-derived outcomes appear favorably affected by MiECC (lung function, acute kidney injury) but the multi-factorial nature of these complications makes difficult to clearly attribute this pattern to a lower degree of inflammation. Overall, the existing body of evidence is in favor of MiECC with respect to standard CPB.

Reagent‐specific underestimation of turoctocog alfa pegol (N8‐GP) clotting activity owing to decelerated activation by thrombin

BackgroundFactor VIII (FVIII) procoagulant activity is commonly assessed by measuring the activated partial thromboplastin time (APTT) to clot formation using one of the many available but differently composed reagents. The majority of APTT reagents also accurately recover the activity of the extended half‐life molecule N‐glycoPEGylated FVIII (N8‐GP; turoctocog alfa pegol), while a few silica‐based reagents give a low recovery.ObjectiveTo identify the cause of N8‐GP activity underestimation in the presence of certain silica‐based APTT reagents.MethodsDevelopment of FVIIIa‐dependent tenase activity and appearance of FVIIIa from N8‐GP and its non‐PEGylated counterpart (N8) were compared using clotting assays, a factor Xa (FXa) activity assay mimic thereof, and thrombin activation time courses.ResultsA strong correlation was demonstrated between clotting and FXa activity assays based on similar recoveries of N8‐GP activity and equal responses to an altered duration of the contact activation phase, validating the latter as a useful clotting assay mimic. Contact activation phase duration influenced, and could even eliminate, N8‐GP activity underestimation. Thrombin‐catalyzed conversion of N8‐GP to FVIIIa was considerably slower than that of N8 despite similar extents of adsorption to silica particles in APTT‐SP, suggesting different modes and/or orientations of adsorption.ConclusionsSome contact activators reduce thrombin's ability to cleave N8‐GP more than native FVIII. Decelerated thrombin activation of N8‐GP is reflected in delayed FVIIIa‐dependent appearance of FXa activity in plasma, in turn leading to prolonged clotting time. This forms the basis for underestimation of N8‐GP activity as measured by one‐stage clotting assay against a FVIII standard.

Prochemerin cleavage by factor XIa links coagulation and inflammation

AbstractChemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

Underestimation of N‐glycoPEGylated factor IX one‐stage clotting activity owing to contact activator‐impaired activation

Unlabelled Box BackgroundIn clinical practice, factor IX (FIX) activity is routinely quantified by measurement of the activated partial thromboplastin time (APTT) in a one‐stage (OS) FIX clotting assay. APTT reagents provide a contact activator and phospholipid surfaces required for triggering and sustaining the plasma clotting process. The large diversity in reagent components is reflected in the variable recovery of nonacog beta pegol (N9‐GP; N‐glycoPEGylated recombinant FIX) activity when assayed against a FIX standard. This variation warrants mechanistic studies and is plausibly attributable to the nature and amount of contact activator. ObjectiveTo identify the cause of the N9‐GP activity underestimation observed with a heterogeneous group of APTT reagents. MethodsExperiments mimicking the clotting phase (omitting the contact activation phase) of the OS assay, complemented by measurements of activated factor XI (FXIa) activity, were performed to characterize and explain the influence of APTT reagents/contact activators on the conversion of N9‐GP and regular FIX (N9) to activated FIX (FIXa). ResultsIn the presence of an intact underestimating APTT reagent or the isolated contact activator, clotting phase activation of N9‐GP proceeded at a reduced rate compared with that of N9. APTT reagent and contact activator negatively affected the activity of FXIa, conceivably as a consequence of FXIa adsorption. Thus, activation of FIX apparently poses a greater steric challenge after polyethylene glycol (PEG) conjugation. ConclusionsSome OS clotting assay contact activators reduce FXIa‐mediated activation of N9‐GP to a larger degree than that of N9, causing underestimation of N9‐GP activity of potential clinical significance.

Hereditary C1 inhibitor deficiency is associated with high spontaneous amidase activity.

Angioedema diagnosis classically targets the complement system (via C1 inhibitor (C1Inh) function and antigenic C4 level) and contact phase activation (via amidase activity). Bradykinin is responsible for angioedema attacks and is produced from contact phase activation secondary to failed C1Inh control. We aimed to compare the diagnostic performances of spontaneous amidase activity and antigenic C4 level in C1Inh hereditary angioedema (C1Inh-HAE) patients. Samples from 185 C1Inh-HAE patients (81 men, 104 women; confirmed by SERPING1 gene mutations) and from 99 blood donors (50 men, 49 women) were tested for C1Inh function, antigenic C4 level and spontaneous amidase activity. In the C1Inh-HAE group, antigenic C4 level was decreased (n=135) and amidase activity was increased (n=181). Receiver operating characteristic analyses showed higher diagnostic performance values for the spontaneous amidase assay compared to those of antigenic C4. The spontaneous amidase activity assay should replace antigenic C4 level testing and should be tested alongside the C1Inh function for both AE screening and follow up of HAE patients.

Prochemerin Cleavage By Factor XIa Links Adipogenesis, Inflammation and Coagulation

Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. Cleavage of chem163S at Lys158 generates chem158K, which has low bioactivity, with subsequent cleavage by the basic carboxypeptidases in plasma, carboxypeptidase N (CPN) or thrombin-activatable CPB2 (also known as thrombin-activatable fibrinolysis inhibitor or TAFI), to produce chem157S, the most active form of chemerin. Inactivation of chem157S to chem155A and smaller forms of chemerin occurs by further proteolytic cleavages.We have developed specific ELISAs for chem163S, 158K, 157S and 155A. Using these chemerin-specific ELISAs, we found that most of the chemerin in normal human plasma is inactive chem163S, while chem158K is the dominant form in synovial fluids of arthritis patients. To identify the physiological activator of chemerin in plasma, we screened proteases from the coagulation, fibrinolytic and complement pathways. The proteases were incubated with purified chem163S protein in buffer and the resultant products were analyzed by MALDI-TOF mass spectrometry. The results showed that Factor XIa (FXIa) can cleave chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. We compared the kinetics of FXIa cleavage of chem163S and FIX, the established physiological FXIa substrate. Peptides from chem163S and FIX containing the FXIa cleavage site were subjected to FXIa cleavage followed by HPLC analysis. The catalytic efficiency (kcat/KM) of FXIa cleavage of FIX was ~3-fold more efficient than chemerin cleavage, but the hydrolysis of chem163S still occurred at a physiologically relevant rate. By substituting Arg162 with Ala in chemerin protein/peptides, we found that processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity.We further investigated FXIa cleavage of chem163 in plasma triggered by kaolin to activate FXI. FXI activity was measured by inclusion of a specific chromogenic substrate, and chemerin levels were measured by the chemerin-specific ELISAs. Contact phase activation of human platelet-poor plasma led to generation of FXIa and cleavage of chem163S, which was abolished in FXI-depleted plasma. Platelets markedly enhanced FXI activation and chem163S cleavage in platelet-rich plasma comparing to platelet-poor plasma. Following contact activation, plasma levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared to a matched control group (90.69±9.6 ng/mL vs. 58.11±2.51 ng/mL, n=8; P<0.01) and inversely correlated with the plasma FXI levels. FXIa, generated upon contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, thus providing a molecular link between coagulation, inflammation, innate immunity and adipogenesis. DisclosuresNo relevant conflicts of interest to declare.

Overestimation of N‐glycoPEGylated factor IX activity in a one‐stage factor IX clotting assay owing to silica‐mediated premature conversion to activated factor IX

Essentials Nonacog beta pegol (N9-GP) activity is overestimated in clot method using silica-based reagents. Mimicking contact activation phase with silica reveals N9-GP activation before recalcification. Localization of N9-GP to silica facilitates activation by factor XIa and plasma kallikrein. Silica-based reagents to be used with caution when monitoring N9-GP therapy using clot method. Background Clinical laboratories routinely quantify factor IX (FIX) activity by measurement of the activated partial thromboplastin time (APTT) in a one-stage (OS) clotting assay. This assay can be performed with any of a plethora of differently composed APTT reagents, giving variable recovery when applied to nonacog beta pegol (N9-GP), an N-glycoPEGylated recombinant FIX. Objective To identify the cause of observed overestimations of N9-GP activity in an OS FIX clotting assay when most APTT reagents containing silica are used as the contact activator, and to elucidate the underlying mechanism. Methods Experiments mimicking the contact activation and clotting phases of the OS assay, combined with the use of plasmas with various deficiencies, were employed to shed light on the unique behavior of N9-GP. Confirmatory activations of N9-GP with purified enzymes and physical adsorption to silica particles were studied, and the influence of free polyethylene glycol (PEG) on these processes was investigated. Results N9-GP, but not native FIX, added to FIX-deficient plasma was prematurely converted to activated FIX (FIXa) during the contact activation phase of the clotting assay. Activated FXI (FXIa) and plasma kallikrein (PK) were responsible for the activation of N9-GP, an event that appeared to require the presence of a silica-containing APTT reagent. PEG-dependent adsorption of N9-GP to silica particles could be demonstrated. Conclusions The PEG moiety mediates colocalization of N9-GP with its activators FXIa and PK on silica surfaces, thereby facilitating premature conversion of N9-GP to FIXa during the contact activation phase, and leading to overestimation of the FIX activity in the OS clotting assay.

C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay.

Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements. The contact phase was reconstituted using the purified components, with C1Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro-Phe-Arg-pNA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics (ROC) were used to calculate the assay's diagnostic performance. The calibration curve was built using C1Inh standard (threshold limit 0.10×10(-3) U, i.e., 0.2pmol), and C1Inh function was quantified in the sample, with a reference interval established based on healthy individuals (n=281; men: 0.61-1.10U/ml, median: 0.85U/ml; women: 0.42-1.08U/ml, median: 0.74U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36U/ml (area under curve [AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61U/ml (AUC: 1; sensitivity: 100.0%; specificity: 100.0%). The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target.

Anex vivoevaluation of blood coagulation and thromboresistance of two extracorporeal circuit coatings with reduced and full heparin dose

Bioactive Carmeda® heparin-coated extracorporeal circuits (ECCs) have been shown to reduce contact phase and coagulation activation during cardiopulmonary bypass (CPB). Heparin coating is therefore effective in safely reducing coagulation during routine CPB. Balance® Biosurface is a new, recently developed biopassive coating containing negatively charged sulphonated polymers. This study sought to compare the clotting activation and thromboresistance of the Balance® (B) circuit with that of the Carmeda® (C) with full-dose systemic heparin (FDH) and reduced-dose systemic heparin (RDH). This ex vivo study set-up comprising 40 experiments consisted of simplified ECC and circulation of freshly donated human blood. RDH and FDH regimens were obtained with 0.5 IU/ml and 1 IU/ml heparin administered to reach target activated clotting times (ACTs) of 250 and 500 s, respectively. The study design comprised four groups: FDH-C, FDH-B, RDH-C and RDH-B (all n = 10). Blood was sampled prior to and during the 2-h CPB. Coagulation activation was assessed (FXIIa, F1.2) and electron microscope scan imaging of oxygenators enabled determination of adhesion scores. With a biopassive compared with bioactive surface, mean ACT was lower, regardless of the heparin regimen applied (P < 0.001), whereas the total heparin dose required to maintain ACT was above target level (P < 0.001). However, FXIIa and F1.2 values were similar in all groups throughout, as were pressure gradients among oxygenators. All groups demonstrated similar adhesion scores following ultrastructural oxygenator assessment. In the absence of surgical-related haemostatic disturbances and based on target ACT levels under reduced- or full-dose heparin, the clotting process was similar to heparin-coated and new sulphonated polymer-coated ECC, both demonstrating similar thromboresistance.

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Influence of the F12-4 C&gt;T polymorphism on hemostatic tests

The common F12 -4 C>T polymorphism significantly regulates plasma levels of FXII, the first element of the intrinsic pathway of coagulation. Due to the robust effects that this pathway has on blood coagulation in vitro, the objective of our study was to evaluate the influence of this polymorphism on different hemostatic tests. We studied 46 hemostatic parameters in 566 participants: 280 patients with mucocutaneous bleeding and 286 controls. The F12 -4T allele, associated with reduced levels of FXII (P < 0.001), also significantly delayed the activated partial thromboplastin time (aPTT) expressed as aPTTr (ratio sample plasma/normal pooled plasma). Thus, both patients and controls carrying the T allele had higher aPTTr than C/C homozygous individuals (P < 0.001). Interestingly, 92% of healthy controls who had prolonged aPTTr carried the F12 -4T allele. Moreover, individuals with the F12 -4T allele also had less thrombin generation (assessed by endogenous thrombin potential, thrombin peak and time to achieve the peak of thrombin) using a test with low tissue factor concentration and explicit contact phase activation. Finally, both patients and controls carrying the F12 -4T allele also displayed significantly lower FIXc and FVIIc levels than C/C individuals (P < 0.01). For all associations except for FVIIc, a gene-dosage effect was observed, and homozygous TT individuals had the farthest values. Our study reveals a significant effect of the F12 -4 C>T polymorphism on hemostatic tests widely used in routine clinical practice.

Contribution of Intrinsic and Extrinsic Coagulation Pathways to Thrombus Formation in the Mouse Carotid Artery.

We studied how the intrinsic and extrinsic coagulation pathways contribute to thrombus formation. Ex vivo experiments were performed with citrated blood re-calcified before perfusion over different thrombogenic substrates. This allowed evaluation of fibrin deposition along with platelet adhesion/aggregation in real time and under relevant shear rates. Under these conditions, human melanoma cells expressing human tissue factor (TF) supported platelet adhesion/aggregation and fibrin formation, but only under relatively low shear rate (200–800 s-1). Anti-TF antibodies markedly inhibited both platelet and fibrin deposition, and no reactivity was observed using the parental cell line not transfected with TF. Blood perfusion over fibrillar collagen type I resulted in platelet adhesion/aggregation and subsequent fibrin deposition even under relatively high shear rate (1500 s-1). Corn trypsin inhibitor (CTI), a specific inhibitor of coagulation factor XIIa, had no effect on platelet adhesion/aggregation but inhibited fibrin deposition, while anti-TF antibodies had no significant effect. Preincubation of blood with prostaglandin E1 or a monoclonal antibody against integrin αIIbβ3 blocked not only thrombus growth on the surface but also fibrin formation. Such a finding indicates that thrombin generation leading to fibrin deposition occurs after platelet activation. Blood perfusion over dermal fibroblast extracellular matrix (F-ECM) from mice expressing human but lacking murine TF resulted in the rapid formation of platelet- and fibrin-rich thrombi at all shear rates tested (up to 1500 s-1). Using this thrombogenic substrate containing both collagen and TF, we found that blockade of the intrinsic coagulation pathway with CTI had a minimal effect on platelet adhesion/aggregation and fibrin formation, while inhibition of the extrinsic coagulation pathway with anti-human TF antibodies reduced thrombus growth and stabilization and abolished fibrin formation. The results of these ex vivo studies demonstrate that the relative contribution of the intrinsic and extrinsic coagulation pathways to fibrin and platelets deposition depends on the composition of the thrombogenic surface exposed to blood flow, and indicate also that CTI and anti-TF antibodies can be used as specific inhibitors of thrombin generation through the intrinsic and extrinsic coagulation pathways, respectively. We then established a model of carotid artery injury induced by ferric chloride to evaluate whether the information obtained under controlled experimental conditions ex vivo can help interpret the mechanisms underlying arterial thrombus formation in vivo. In this model, all control C57BL6 mice exhibited a stable carotid artery occlusion within a predictable time frame. To obtain a quantitative parameter of the progression of thrombosis we calculated a flow index. This represents the ratio between the volume of blood that flew through the injured artery from the induction of the lesion to occlusion (or to the end of the predetermined 30 min observation period) and the volume of blood that during the same period of time should have flown through the artery if the initial (pre-injury) flow rate had been maintained. We found that blocking the intrinsic coagulation pathway with CTI injected through the jugular vein significantly delayed, and in most cases prevented, the formation of a stable occlusion, resulting in a significantly altered flow index. The injection of antibodies against human TF, on the other hand, resulted in a trend towards thrombus destabilization, but in most cases the artery still occluded and the flow index was less significantly altered that with CTI. Of note, CTI at a dose that inhibited carotid artery occlusion had no effect on the tail bleeding time. Co-administration of CTI and anti-TF antibodies showed a cooperative effect across the tested concentration range. In conclusion, extrinsic and intrinsic coagulation pathways have complementary roles in thrombus formation and stabilization, and the specific contribution of each depends upon the nature of the thrombogenic surface, i.e. of the causative lesion. The marked effect of factor XIIa inhibition in preventing carotid artery occlusion suggests that a functional link between contact phase activation and tissue factor pathway leading to thrombin generation may be operative under defined conditions in vivo.

The effect of gas plasma modification on platelet and contact phase activation processes

Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O 2, Ar, N 2 and NH 3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.

Anti-hemostatic Effects of a Serpin from the Saliva of the Tick Ixodes ricinus

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.