Siw Frebelius

Rapid radioimmunoassay of human fibrinopeptide A — Removal of cross-reacting fibrinogen with bentonite

Abstract Bentonite was used for the rapid removal of cross-reacting fibrinogen from plasma before radioimmunoassay of human fibrinopeptide A (FPA). The recovery of added fibrinopeptide was 85 – 90%. Normal FPA values in plasma were 1.4 ng/ml (SD 0.9). Incubation times and temperatures were evaluated to select optimal conditions. The reduction of time for plasma processing and assay to less than two hours did not reduce the sensitivity (detection limit 0.019 ng/ml) and greatly increases the clinical applicability of the FPA assay.

Thrombin Inhibition by Antithrombin III on the Subendothelium Is Explained by the Isoform ATβ

Balloon injury of the rabbit aorta results in thrombin coagulant activity on the injured vessel wall that causes fibrin formation. The anticoagulant activity of both the intact and injured vessel wall has been partly explained by glycosaminoglycans with heparin-like activity that augment that activity of antithrombin III (AT). AT exists in two isoforms, alpha and beta, AT beta, which constitutes only 5% to 10% of AT in plasma, lacks one carbohydrate side chain, has higher affinity for glycosaminoglycans, and associates more readily with the subendothelium. This study evaluated whether AT can inhibit thrombin on the injured vessel wall and, if so, whether one of the isoforms is more effective then the other. The two isoforms were isolated from human plasma by heparin-Sepharose chromatography, and the purity was investigated by isoelectric focusing and crossed immunoelectrophoresis. Rabbits were subjected to balloon injury of the aorta; 3 hours after injury the aorta was excised. Thrombin coagulant activity on the aorta was measured by exposure to fibrinogen and thereafter by measuring the generation of fibrinopeptide A. Injured animals were treated with AT, AT alpha, or AT beta and were compared with control animals. AT was demonstrated on the injured vessel wall by using an immunohistochemical method. Animals receiving crude AT had significantly lower amounts of thrombin coagulant activity on the injured aortic wall than control animals, but AT alpha at a comparable dose had no effect. AT beta was given in the same dose as crude AT and also at a dose (10%) proportional to its presence in plasma. Animals receiving AT beta had significantly lower values of thrombin on the injured aortic wall than control animals. We conclude that the inhibitory effect of AT on thrombin coagulant activity on the injured vessel wall in explained by its AT beta content.

Antithrombin III inhibits thrombin-induced proliferation in human arterial smooth muscle cells.

Thrombin has attracted increasing attention as a possible mitogen for vascular smooth muscle cells in lesion development both after vascular injury and in atherogenesis. In this study, the ability of antithrombin III to inhibit alpha-thrombin-induced DNA synthesis and cell proliferation in human arterial smooth muscle cells was analyzed. We demonstrate a concentration-dependent initiation of DNA synthesis and cell proliferation by alpha-thrombin. This effect was abolished when complex formation with antithrombin III was allowed before thrombin was added to the cell cultures. Addition of alpha-thrombin and antithrombin III simultaneously at the beginning of the incubation period also resulted in an inhibition of thrombin-induced DNA synthesis, but to a lower degree. The inhibitory activity of antithrombin III was enhanced in the presence of heparin, which on its own had no inhibitory effect on thrombin-induced DNA synthesis. In contrast, the mitogenic activity of alpha-thrombin could be inhibited by heparin in the presence of low concentrations of serum. This inhibition was dependent on the presence of antithrombin III in serum, since heparin lacked effect if antithrombin III was depleted from serum by immunoaffinity chromatography. Analysis of the enzymatic activity of thrombin showed that the influence on catalytic activity of thrombin corresponded to the mitogenic activity of thrombin in the presence of heparin, antithrombin III, and serum. The results suggest that the mitogenic activity of thrombin is regulated by antithrombin III. Therefore, antithrombin III may serve dual functions by inhibiting thrombin in the coagulation cascade and by neutralizing its growth-promoting effects on vascular smooth muscle cells.

Proteolytically active ADAM10 and ADAM17 carried on membrane microvesicles in human abdominal aortic aneurysms

The intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) has been suggested to damage the underlying aortic wall, but previous work found scant activity of soluble proteases in the abluminal layer of the ILT, adjacent to the aneurysm. We hypothesised that transmembrane proteases carried by membrane microvesicles (MV) from dying cells remain active in the abluminal ILT. ILTs and AAA segments collected from 21 patients during surgical repair were assayed for two major transmembrane proteases, ADAM10 (a disintegrin and metalloprotease-10) and ADAM17. We also exposed cultured cells to tobacco smoke and assessed ADAM10 and ADAM17 expression and release on MVs. Immunohistochemistry showed abundant ADAM10 and ADAM17 protein in the ILT and underlying aneurysmal aorta. Domain-specific antibodies indicated both transmembrane and shed ADAM17. Importantly, ADAM10 and ADAM 17 in the abluminal ILT were enzymatically active. Electron microscopy of abluminal ILT and aortic wall showed MVs with ADAM10 and ADAM17. By flow cytometry, ADAM-positive microvesicles from abluminal ILT carried the neutrophil marker CD66, but not the platelet marker CD61. Cultured HL60 neutrophils exposed to tobacco smoke extract showed increased ADAM10 and ADAM17 content, cleavage of these molecules into active forms, and release of MVs carrying mature ADAM10 and detectable ADAM17. In conclusion, our results implicate persistent, enzymatically active ADAMs on MVs in the abluminal ILT, adjacent to the aneurysmal wall. The production of ADAM10- and ADAM17-positive MVs from smoke-exposed neutrophils provides a novel molecular mechanism for the vastly accelerated risk of AAA in smokers.

The influence of direct and antithrombin-dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin

INTRODUCTION Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin. MATERIALS AND METHODS We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). RESULTS All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively. CONCLUSIONS All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.

Heparin cofactor II significance for the inhibition of thrombin at the injured vessel wall

The thrombin inhibitory role of antithrombin III (ATIII) and heparin cofactor II (HCII) was studied in vitro using intact and injured rabbit aortae. When intact vessels were loaded with thrombin and then exposed to either heat defibrinogenated human plasma (HDHP) or ATIII the same degree of thrombin inhibition was achieved demonstrating that ATIII was the only plasma component involved in thrombin inhibition on the intact vessel wall. When the media of the vessel wall was loaded with thrombin and then exposed to ATIII or HCII a significantly higher thrombin activity remained on the surface than when it was exposed to defibrinogenated plasma. A mixture of ATIII and HCII resulted in a greater inhibition of thrombin than ATIII or HCII alone. It is concluded that, contrary to what happens on the endothelium, HCII and ATIII inhibit additively thrombin on the injured vessel wall. HCII thus plays an essential role for the inhibition of thrombin at the injured vessel wall. It is also concluded that an additional plasma component participates in thrombin inhibition on the media but its contribution is negligible as compared with ATIII or HCII.

Inhibition of neointimal hyperplasia by a specific thrombin inhibitor

Objective—Restenosis secondary to neointimal hyperplasia remains the major limiting factor after vascular interventions. Thrombin generated in high concentrations at the site of vascular injury plays a central role in thrombosis and hemostasis. Thrombin has also been implicated as a mitogen for smooth muscle cell proliferation that contributes to restenosis. This study was designed to determine the effects of a specific thrombin inhibitor on neointimal hyperplasia after balloon injury in a rat carotid artery model. Design—A total of 47 male Sprague–Dawley rats were divided into five groups. All groups underwent balloon injury of the left carotid artery. A specific thrombin inhibitor, inogatran, was given in four different regimens: low and high dose injections, short‐term infusion for 3 h, and long‐term infusion for 1 week. After 2 weeks the animals were killed and the carotid neointima/media area ratio and the luminal narrowing were calculated. Results—All treatments significantly reduced the neointimal hyperplasia. Inogatran given as a long‐term infusion for 1 week had the lowest neointima/media ratio compared with the other groups. The percentage of lumen narrowing was also significantly lower in all treatment groups compared with the control group. Conclusion—A specific direct thrombin inhibitor, inogatran, reduces neointimal hyperplasia after arterial injury in rats. A more prolonged administration of the thrombin inhibitor gave a further reduction of the neointimal hyperplasia. It seems that inhibition of thrombin activity is not only important early after injury, but also later. This could have clinical implications in the treatment of restenosis and needs to be further evaluated.

Neutrophil Elastase-Derived Fibrin Degradation Products Indicate Presence of Abdominal Aortic Aneurysms and Correlate with Intraluminal Thrombus Volume

BACKGROUND The intraluminal thrombi (ILT) of abdominal aortic aneurysms (AAA) contain neutrophils, which can secrete elastase. We evaluated whether plasma neutrophil elastase-derived cross-linked fibrin degradation products (E-XDP) could reveal the presence, size and mechanical stress of AAAs and its ILTs. METHODS E-XDP and D-dimer were measured in plasma from 37 male patients with AAA and 42 male controls. The ILT volumes of the AAAs and any coexisting aneurysms could be measured in 29 patients and finite element analysis was performed to estimate mechanical stress of the ILT. E-XDP, neutrophil elastase and neutrophil marker CD66acd were evaluated in aortic tissue with immunohistochemistry (IHC). The association between ILT volume and E-XDP was validated in a separate cohort (n = 51). RESULTS E-XDP levels were elevated in patients with AAA compared with controls (p = 5.8e-13), indicated AAA with 98% sensitivity, 86% specificity and increased with presence of coexisting aneurysms. The association between AAA and increased E-XDP was independent of smoking, comorbidities and medication. E-XDP correlated with volume of all ILTs (r = 0.76, p = 4.5e-06), mean ILT stress (r = 0.46, p = 0.013) and the volume of the AAA-associated ILT (r = 0.64, p = 0.00017). E-XDP correlated stronger with ILT volume compared with D-dimer (r = 0.76 vs. r = 0.64, p = 0.018). The correlation between E-XDP and ILT volume was validated in the separate cohort (r = 0.53, p = 7.6e-05). IHC revealed E-XDP expression in the ILT, spatially related to neutrophil elastase and neutrophils. CONCLUSION E-XDP is a marker of the presence of AAA and coexisting aneurysms as well as the volume and mechanical stress of the ILT.