Raymund Machovich

Mechanism of action of heparin through thrombin on blood coagulation

It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, T., Blaskó, Gy. and Pálos, L. (1975) Biochim. Biophys. Acta 379, 193-200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.

Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-κB as a signal transducer

The haemorrhagic transformation in ischemic stroke involves disruption of the integrity of the microvascular beds, partially based on the action of matrix metalloproteinases (MMPs).The objective of the present study was to evaluate the contribution of microvascular endothelial cells from human brain (HBECs) to MMPs’ expression and regulation under conditions relevant to brain ischemia. MMPs and their inhibitors were examined with zymography, Western-blotting, ELISA and MMP-activity assay in cultured HBECs. Four-hour hypoxia (pO2 =60 mmHg) elevated the level of MMP-9 in the supernatant of the HBECs and this early response required collagen-matrix.Active oxygen species sustained the increased MMP-9 activity for at least 24 h. In the post-hypoxic period 20 µmol/L H2O2 caused a 6-fold increase in the specific activity of MMP-9 over the nor-moxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-κB, a redox-state sensitive transcription factor, was evaluat-ed with immunofluorescence confocal microscopy and immu-noblotting of nuclear and cytoplasmic extracts. The oxidative stress-dependent MMP-9 induction was accompanied by a sig-nificant increase in the NF-? B localized in the nuclei and these responses were blunted with a proteasome inhibitor (MG132). Consequently, according to our in vitro data HBECs are a source of MMP-9, which is under the control of triggers relevant to the ischemic/reperfused brain (reactive oxygen species, thrombus and inflammation related proteases) and this regulation is par-tially based on NF-? B activation. The reported regulation of endothelium-derived MMP-9 supports its potential involve-ment in the post-hypoxic disturbances of the cerebral micro-circulation.

Action of heparin on thrombin-antithrombin reaction

Thrombin partially purified from bovine plasma can be inactivated at 60 degress C. In the presence of 10 units of heparin the extent of inactivation decreases. When thrombin and heparin are mixed and incubated for 5 min at 0 degrees C before gel filtration on Sephadex G-200, thrombin with heparin is eluted prior to either thrombin or heparin laone. These data suggest a complex formation between thrombin and heparin. Immobilized heparin binds thrombin. The enzyme can be eluted with 0.05 M Tris-HCl buffer, pH 7.3, containing an ion mixture of Na+, K+ and Ca2+ at 73, 3 and 11 mM, respectively, at 0 degrees C and with 0.05 M Tris-HCl buffer, pH 7.3, containing 0.5 M NaCl at 20 degrees C. During the same chromatographic procedure, antithrombin-III (heparin cofactor) partially purified from human plasma is eluted with 0.05 M Tris-HCl buffer, pH 7.3, at 0 degrees C as well as 20 degrees C. Although, as described in the literature, heparin binds to antithrombin, our findings suggest another possibility, i.e. that the binding of heparin to thrombin induces a conformational change in the enzyme facilitating a complex formation between thrombin and antithrombin-III.

Hindered dissolution of fibrin formed under mechanical stress

See also Weisel JW. Stressed fibrin lysis. This issue, pp 977–8.

Lytic Resistance of Fibrin Containing Red Blood Cells

Objective—Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. Methods and Results—If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein–labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. Conclusion—RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

The elastase-mediated pathway of fibrinolysis

Plasmin and elastase degrade fibrin and inhibit the blood coagulation system by degrading key proteins. Elastase can facilitate plasmin expression via an alternative pathway of plasminogen activation. Elastase modifies plasminogen to yield a zymogen that is a better substrate for activators than native plasminogen. Furthermore, elastase inactivates the inhibitor system of plasmin and plasminogen activators without affecting plasmin and plasminogen activators. While plasmin activity develops from a blood zymogen as a consequence of activators synthesized and secreted by endothelium and possibly other cells, elastase is secreted in an active form primarily by polymorphonuclear leukocytes. Plasmin and elastase may play mutual roles in thrombolysis, inflammation, and tumour invasion and metastasis.

Identification of Thrombin Receptors in Rat Brain Capillary Endothelial Cells

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombins cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.

Contraction of Human Brain Endothelial Cells Induced by Thrombogenic and Fibrinolytic Factors An In Vitro Cell Culture Model

BACKGROUND AND PURPOSE Vasogenic brain edema is a frequent complication of ischemic stroke. The mechanism of the blood-brain barrier opening that underlies the edema formation is poorly understood. In the present study we examined the response of endothelial cells cultured from adult human brain to thrombogenic and fibrinolytic factors that possibly accumulate in the occluded vascular segments in ischemic stroke. METHODS The changes in the morphology of cultured human brain microvascular endothelial cells were observed by phase-contrast light microscopy and quantified with computerized morphometry. RESULTS Active proteases (eg, thrombin, plasmin, urokinase) as well as heparin and protamine, but not fibrinogen and antithrombin III, produced significant changes in endothelial cell morphology. Two shape patterns of contraction were observed: protamine treatment resulted in rounded cells with a decrease in both cell perimeter and area, whereas all other agents induced spiderlike cell morphology with increased perimeter and reduced area. The rate of contraction was dose dependent, and at comparable enzyme concentrations plasmin produced faster contraction than thrombin. The observed changes were reversed 3 hours after abrogating the treatment. CONCLUSIONS In an in vitro model we have demonstrated that factors involved in thrombus formation and dissolution induce endothelial cell contraction, which could affect focally the permeability of the blood-brain barrier by opening paracellular avenues between endothelial cells in vivo. Thus, the genesis of brain edema in thromboembolic stroke or occasionally during fibrinolytic therapy can be attributed in part to the contact of these factors with the microvascular endothelium.

DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment

In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10(-9) cm(2). Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

Perturbation of the integrity of the blood-brain barrier by fibrinolytic enzymes

The action of fibrinolytic enzymes (plasmin, miniplasmin, neutrophil leukocyte elastase) on the blood-brain barrier is investigated. The binding and the effects of the fibrinolytic enzymes are studied in the first subcultivation of human brain capillary endothelial cells. 125I-labeled plasmin, miniplasmin and neutrophil leukocyte elastase bind to confluent monolayers of cultured endothelial cells with dissociation constants of 1 × 10-8 mol/l, 4.8 × 10-7 mol/l and 1.8 × 10-8 mol/l, respectively, and the number of binding sites varies between 2.3 × 105 and 7.5 × 106 per cell. Following treatment of the cultured cells with purified and active-site titrated proteases, the changes in morphology of individual cells are analyzed with computerized morphometry. At low concentrations (in nanomolar range) all studied fibrinolytic proteases induce reduction of the cell area; the minimal size is achieved in 20–80 min after the application of an enzyme and the effect is completely reversed in 15 min after its removal. A possible in-vivo consequence of these in-vitro findings is studied in an organ-perfusion model: rat hemisphere is perfused with a protease solution followed by a circulating phase-borne tracer (horse-radish peroxidase). In perfused rat hemisphere, the fibrinolytic enzymes open the blood-brain barrier to the circulation-borne tracer. These results support the concept that fibrinolytic enzymes interact with the brain microvascular endothelium and thus affect the integrity of the blood-brain barrier through active cell contraction.