In-vitro assessment of the effect of dabigatran on thrombosis of adult and neonatal plasma: comparisons using thromboelastography and microscopic visualization of fibrin clot structure.

: Thromboelastography (TEG) is a global assay used for evaluating features of clot formation in vitro. Dabigatran is a reversible direct inhibitor of thrombin that has not been studied in neonates using a sophisticated global assay, such as TEG. Neonatal hemostasis differs from adult hemostasis in both quantitative and qualitative characteristics. Our aim was to compare the TEG clotting profile of neonatal and adult platelet-poor plasma when exposed to different concentrations of dabigatran. We used commercially collected adult pooled plasma and neonatal cord blood collected from placentas of healthy full term newborns. Platelet-poor plasma was isolated, pooled, and frozen. Prior to experiment, plasma was thawed and filtered. A reaction mixture of CaCl2, corn trypsin inhibitor, tissue factor, and dabigatran in imidazole buffer was mixed with plasma in a TEG cup. Time to clot initiation (R-time), speed of clot strengthening (α-angle), and maximum clot strength (maximal amplitude) were measured. Scanning electron microscopy was performed to evaluate fibrin clot structure. Without dabigatran, there was no significant difference in TEG measurements between neonatal and adult samples. However, neonatal plasma clotting with dabigatran had slower onset, slower speed, and weaker clots that were more porous with thicker fibers, compared with adult plasma clotting. Thus, neonatal plasma may be more sensitive to dabigatran as assessed by our in-vitro TEG study.

Inhibition of plasmin generation in plasma by heparin, low molecular weight heparin, and a covalent antithrombin-heparin complex.

: The clinical limitations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) led to the development of an antithrombin-heparin covalent complex (ATH), which displays superior anticoagulant abilities compared with UFH. A recent study investigating its interaction with fibrinolysis showed that ATH inhibited free and fibrin bound plasmin and decreased plasmin generation on fibrin clots. These studies were conducted using purified components and did not elucidate the interaction of ATH with plasmin in the presence of its natural inhibitors α2-antiplasmin (α2-AP) and α2-macroglobulin (α2-M). The aim of this study was to determine the effects of ATH, UFH, and LMWH on plasmin generation in plasma, under more physiological conditions. Plasmin generation in plasma in the absence and presence of anticoagulants was initiated by tissue plasminogen activator and soluble fibrin fragments, and plasmin and plasmin-α2-M complexes generated over time were quantified chromogenically. Generation of plasmin-α2-AP complexes and consumption of plasminogen were quantified by ELISA. Plasmin generation was decreased in the presence of UFH and ATH, whereas LMWH had no effect. Neither plasminogen consumption nor generation of plasmin-α2-AP complexes were affected by UFH or ATH. However, plasmin-α2-M complexes were slightly reduced by ATH suggesting that ATH may be able to compete with α2-M for plasmin. Plasmin generation may be mildly inhibited by heparin-based anticoagulants; however, heparin-catalyzed antithrombin activity is not a major inhibitor of plasmin, as compared to its natural inhibitors α2-AP and α2-M. This adds to our understanding of ATH mechanisms of action and aids in its development for clinical use.

Surface modification of poly(dimethylsiloxane) with a covalent antithrombin-heparin complex for the prevention of thrombosis: use of polydopamine as bonding agent.

A modified poly(dimethyl siloxane) (PDMS) material is under development for use in an extracorporeal microfluidic blood oxygenator designed as an artificial placenta to treat newborn infants suffering from severe respiratory insufficiency. To prevent thrombosis triggered by blood-material contact, an antithrombin-heparin (ATH) covalent complex was coated on PDMS surface using polydopamine (PDA) as a "bioglue". Experiments using radiolabelled ATH showed that the ATH coating on PDA-modified PDMS remained substantially intact after incubation in plasma, 2% SDS solution, or whole blood over a three day period. The anticoagulant activity of the ATH-modified surfaces was also demonstrated: in contact with plasma the ATH-coated PDMS was shown to bind antithrombin (AT) selectively from plasma and to inhibit clotting factor Xa. It is concluded that modification of PDMS with polydopamine and ATH shows promise as a means of improving the blood compatibility of PDMS and hence of the oxygenator device.

Interactions of heparin and a covalently-linked antithrombin-heparin complex with components of the fibrinolytic system.

Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with limitations, such as the inability to inhibit surface bound coagulation factors. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties compared with UFH. Advantages of ATH include enhanced inhibition of surface-bound coagulation enzymes and the ability to reduce the overall size and mass of clots in vivo. The interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Our study utilised discontinuous second order rate constant (k2) assays to compare the rates of inhibition of free and fibrin-associated plasmin by AT+UFH vs ATH. Additionally, we evaluated the effects of AT+UFH and ATH on plasmin generation in the presence of fibrin. The k2 values for inhibition of plasmin were 5.74 ± 0.28 x 106 M⁻¹ min⁻¹ and 6.39 ± 0.59 x 106 M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. In the presence of fibrin, the k2 values decreased to 1.45 ± 0.10 x 106 M⁻¹ min⁻¹ and 3.07 ± 0.19 x 106 M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. Therefore, protection of plasmin by fibrin was observed for both inhibitors; however, ATH demonstrated superior inhibition of fibrin-associated plasmin. Rates of plasmin generation were also decreased by both inhibitors, with ATH causing the greatest reduction (approx. 38-fold). Nonetheless, rates of plasmin inhibition were 2-3 orders of magnitude lower than for thrombin, and in a plasma-based clot lysis assay ATH significantly inhibited clot formation but had little impact on clot lysis. Cumulatively, these data may indicate that, relative to coagulant enzymes, the fibrinolytic system is spared from inhibition by both AT+UFH and ATH, limiting reduction in fibrinolytic potential during anticoagulant therapy.

Comparison of N-linked glycosylation of protein C in newborns and adults.

Protein C (PC) is a major anticoagulant that stems the propagation of thrombin. The activated form of PC (APC), in association with the cofactor protein S, proteolytically converts activated coagulation factors VIIIa and Va into inactive forms. Studies have shown that forms of PC that contain 3N-linked glycans (beta-PC) are functionally distinct from the fully glycosylated 4-glycan type (alpha-PC). Since some findings have also hinted at qualitative differences in PC from newborns and adults, we decided to determine the relative constitution of glycoforms in these age groups. Subtypes of PC in newborn and adult plasmas were distinguished by SDS polyacrylamide electrophoresis and Western blotting, followed by immunological analysis. Newborns were found to have alpha-PC/beta-PC mole ratios of 8.8:1, compared to 2.3:1 in adults. PC was also isolated by immunoaffinity chromatography from newborn and adult plasmas. Glycans were released by protease treatment and studied by mass spectrometry. Results from glycan analysis showed a small range of glycan structures in both age groups. No clear differences were noted between newborn and adult PC microheterogeneity in glycan structures (branching). We conclude that newborns have important differences in PC macroheterogeneity in glycoform content relative to adults. This age-dependent glycosylation variation may have implications in management of PC function in vivo.

Determination of alpha-2-macroglobulin complexes by a new immuno-activity assay.

INTRODUCTION: Alpha-2-macroglobulin (α(2)M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various α(2)M complexes may be useful to define markers for the status of different hemostatic components. We present proof of principle for a novel assay to quantitatively measure α(2)M in complex with a variety of hemostatic factors. MATERIALS AND METHODS: The assay makes use of the fact that α(2)M entraps proteases within a molecular "cage", leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-α(2)M antibodies were used to isolate the complexes from buffer or plasma, followed by detection of specific proteases with chromogenic substrates. Macromolecular inhibitors were added to eliminate signal from any unbound proteases. RESULTS: Calibration curves constructed with purified protease-α(2)M complexes were sigmoidal in nature, as is typical with immuno-assays. The specificity of signal production was confirmed with inhibitors that target either free protease, or both free and α(2)M-bound protease. The detection range of the assay was dependent on the protease being measured, and the surrounding matrix. Interference in detection of complexes in plasma was found to be caused, in part, by free α(2)M. Thrombin-α(2)M complexes were quantified in adult and newborn plasma following induction of thrombin generation and found to be significantly higher in adults, likely due to higher prothrombin levels. CONCLUSIONS: This assay provides a versatile platform method for quantification of multiple protease-α(2)M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications.

Anticoagulant mechanisms of covalent antithrombin-heparin investigated by thrombelastography. Comparison with unfractionated heparin and low-molecular-weight heparin.

We have developed an antithrombin-heparin covalent complex (ATH) which inhibits coagulation enzymes by two mechanisms: directly, or by catalytic activation of plasma antithrombin (AT). Anticoagulation by ATH was compared to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) using a blood-based, tissue factor (TF)-activated thrombelastography (TEG) assay. Simplified TEG assays with plasma or purified plasma components were used to determine the contribution of the direct and catalytic mechanisms to ATH efficacy. Low anti-Xa concentrations of UFH inhibited clot formation significantly more than equivalent concentrations of ATH or LMWH in blood and plasma. ATH had reduced ability to catalyse AT-mediated thrombin (IIa) inhibition compared to UFH. However, at high anti-Xa concentrations, ATH had similar anticoagulant activity to UFH. ATH and non-covalent AT+UFH directly inhibited clotting to a similar degree in AT-deficient plasma. IIa-ATH complexes, which are limited to catalytic inhibition, displayed impaired anticoagulation compared to free ATH, and the magnitude of this effect increased significantly as anticoagulant concentration increased. Kinetic experiments indicated that the rate of reaction of AT with IIa is lower when catalysed by ATH versus UFH. In conclusion, at low anti-Xa doses catalytic inhibition is the primary mechanism of ATH anticoagulation, and the catalytic potential of ATH is reduced relative to UFH. However, the direct inhibitory activity of ATH is comparable to non-covalent AT+UFH, and at high anti-Xa doses the direct inhibitory activity of ATH may play a larger role in anticoagulation.

Treatment of endothelium with the chemotherapy agent vincristine affects activated protein C generation to a greater degree in newborn plasma than in adult plasma.

INTRODUCTION: Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and alpha(2)macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. MATERIALS AND METHODS: APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-alpha(1)AT were assayed by ELISA. APC-alpha(2)M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. RESULTS: Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-alpha(1) AT and APC-alpha(2) M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. CONCLUSIONS: The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.

Identification of small molecule chemical inhibitors of the collagen-specific chaperone Hsp47.

Hsp47 is a collagen-specific molecular chaperone whose activity has been implicated in the pathogenesis of fibrotic diseases. Here, we describe the development of an assay for screening libraries of chemical compounds for inhibitors of Hsp47. A preliminary screen of 2080 compounds identified four that demonstrated inhibitory activity against Hsp47 in vitro, with IC(50) values ranging from 3 to 27 muM. Compounds identified through this method may provide the basis for development of novel antifibrotic therapeutics.