Variability in exposure of epitope G40-R43 of domain i in commercial anti-beta2-glycoprotein I IgG ELISAs.

BACKGROUND: A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β2glycoprotein I (β2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β2GPI opens up, thereby exposing G40-R43. OBJECTIVES: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. METHODS: Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β2GPI irrespective of its conformation. These antibodies were tested in commercial anti-β2GPI assays (A-E). RESULTS: In assay A, both antibodies showed equal reactivity towards β2GPI, indicating that all the β2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. CONCLUSIONS: Exposure of epitope G40-R43 on β2GPI is highly variable between commercial anti-β2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43

Variability in exposure of epitope G40-R43 of domain in manual and automated commercial anti-beta2-glycopreotein I IgG immunoassays

C

Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-Beta2-Glycoprotein I IgG ELISAs

A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-\u3b22glycoprotein I (\u3b22GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces \u3b22GPI opens up, thereby exposing G40-R43. OBJECTIVES: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. METHODS: Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes \u3b22GPI irrespective of its conformation. These antibodies were tested in commercial anti-\u3b22GPI assays (A-E). RESULTS: In assay A, both antibodies showed equal reactivity towards \u3b22GPI, indicating that all the \u3b22GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. CONCLUSIONS: Exposure of epitope G40-R43 on \u3b22GPI is highly variable between commercial anti-\u3b22GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a ‘cone and base principle’-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate

Differences in the mechanism of blood clot formation and nanostructure in infants and children compared with adults

Introduction: Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. Materials and methods: We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. Results: Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. Conclusions: This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults

Clinical and serological characteristics of cohort 2 (172 patients).

<p>Clinical and serological characteristics of cohort 2 (172 patients).</p

Reactivity of patient samples of cohort 1 in two different anti-β₂GPI assays.

<p>20 selected patient samples, with the indicated characteristics, were tested for reactivity against coated β2GPI in assay A and assay B. Samples were considered strong positive (+++), positive (++) and weak positive (+) according to the recommendations of the manufacturers.</p>1<p>Arterial or venous thrombosis.</p>2<p>Activated partial thromboplastin time/<b><i>dilute Russell's Viper Venom time.</i></b></p>3<p>Antibody reactivity against epitope G40-R43 of domain I of β₂GPI, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071402#pone.0071402-deLaat1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071402#pone.0071402-Iverson1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071402#pone.0071402-Ioannou1" target="_blank">[5]</a>.</p