Interaction of thrombin with heparin cofactor II and antithrombin III on prostacyclin production by cultured endothelial cells

To investigate the physiologic function of heparin cofactor II (HCII), endothelial cells from human umbilical vein were incubated in vitro for 20 min with 0.5 NIH U/ml thrombin in the presence of HCII or antithrombin III (ATIII), and prostacyclin production determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Although ATIII at 20 mInh.U/ml slightly but significantly inhibited thrombin-induced prostacyclin production, neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) at 1 U/ml accelerated the inhibitory effect of ATIII. HCII at 10 and 20 mInh.U/ml did not decrease thrombin stimulation of prostacyclin production in the presence or absence of UFH or LMWH. However, HCII caused a marked decrease in the thrombin-stimulated prostacyclin prostacyclin production in the presence of 2 mg/ml dermatan sulfate (DS). The significant inhibition by HCII occurred when the DS concentrations were 0.2 microgram/ml and higher. From these results we suggest that HCII may prevent a prostacyclin-induced inhibition of platelet aggregation for hemostasis when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of DS.     

Interaction of abnormal antithrombin-III Toyama with cultured porcine aortic endothelial cells

The abnormal Antithrombin-III (AT-III) Toyama has no ability to interact with heparin in the mast cells or heparin-like glycosaminoglycans on the surface of vascular endothelial cells, so that it has low heparin cofactor activity. In the present study, we have measured the binding capacity of 125I-labeled AT-III to cultured porcine aortic endothelial cells and estimated the inactivation of thrombin by AT-III in the presence of endothelial cells. The abnormal AT-III Toyama had low binding capacity to endothelial cells and had lower heparin cofactor antithrombin activity in the presence of endothelial cells than that of normal AT-III. Our results suggested that heparin-like glycosaminoglycans on endothelial cells play as important a role as heparin does in the regulation of antithrombin activity.     

Thrombin generation and formation of thrombin-antithrombin III complexes in congenital antithrombin III deficiency

Two families with quantitative congenital AT III deficiency and a high incidence of thromboembolism are reported. In two unrelated patients (one from each family) thrombin generation in whole blood occured more rapidly than in the control, as demonstrated by the kinetics of prothrombin consumption, AT III disappearance and thrombin-antithrombin III complexes formation. Similar results were obtained in plasma and can be experimentally reproduced with a plasma depleted of AT III by immunoadsorption using a rabbit anti-AT III antiserum. The addition of purified AT III in vitro leads to a complete correction of the abnormalities when the level of AT III is greater than 0.8 unit/ml.     

Comparative study of antithrombin III.protease complex metabolism by fibroblasts and vascular endothelial cells

125I-labeled human antithrombin III (125I-AT III).protease complexes are specifically bound to both cultured human skin fibroblast (HSF) cells and adult bovine aortic endothelial (ABAE) cells; however, there is a significant difference in the rate and degree of metabolism of the complexes by these two cell types. HSF cells appear to internalize the complexes at a rate of about 2.5 pmole/1 X 10(6) cells/h and subsequently degrade them at a rate of 0.6 pmole/1 X 10(6) cells/h. ABAE cells internalize and degrade the complexes at rates approximately 100 and 30 times lower, respectively. Neither cell type interacts with free 125I-AT III but only with its combined form with either thrombin or trypsin. These data indicate the major role of HSF cells in the removal of AT III.protease complexes from extravascular spaces in the body, in contrast to the inert vascular surface with regard to AT III.protease complexes provided by the vascular endothelium.     

Chloroquine and primary amines inhibit the internalization of antithrombin III. Trypsin complex in cultured cells

Bovine corneal endothelial (BCE) cells have been shown to specifically bind, internalize and degrade antithrombin III (AT III) protease complexes as well as thrombin. Previous studies have indicated that chloroquine has no effect on the internalization of thrombin or other cell surface-bound ligands, but it inhibits their subsequent degradation. In contrast, the present study demonstrates the unique inhibitory effect of chloroquine on the internalization of ¹²⁵I-AT III. trypsin complex by BCE cultures. Similarly, the primary amines, monodansylcadaverine and methylamine, inhibit the internalization of ¹²⁵I-AT III . trypsin complex, but not the internalization of ¹²⁵I-thrombin. The various amines used in this study revealed:

(1) differences in the process of cellular binding and internalization between AT III . protease complex and thrombin, although the degradation of both internalized ligands proceed in an analogous manner; and (2) the unique sensitivity to chloroquine of ¹²⁵I-AT III . trypsin complex internalization by cultured cells. These results might indicate that AT III . protease complexes are internalized via a distinct receptor and/or a different mechanism from thrombin.     

Prophylactic antithrombin III administration during pregnancy immediately reduces the thrombin hyperactivity of congenital antithrombin III deficiency by forming thrombin-antithrombin III complexes.

We examined the changes of haemostatic molecular markers after antithrombin III (AT III) administration in a 22-year-old woman with congenital AT III deficiency in the third trimester of pregnancy who did not have thrombosis. Various markers including fibrinopeptide A (FPA), thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), plasmin-alpha 2antiplasmin, D-dimer, beta-thromboglobulin, and platelet factor 4 were measured before and just after 3,000 U of AT III concentrate, which was given three times per week from the 34 week of pregnancy until delivery. Just after AT III administration, F1 + 2 and FPA levels decreased on most occasions, while TAT sometimes increased. Plasma FPA levels were markedly decreased on all 8 occasions when the plasma FPA levels was above 2.0 ng/ml before AT III administration. Plasma FPA levels were always greater than or equal to 6.4 ng/ml before AT III administration on the 4 occasions when TAT increased to above 115%. The changes of plasma F1 + 2 levels were significantly correlated with the AT III level. These results suggest that prophylactic AT III administration in the third trimester immediately inactivates intravascular thrombin to form TAT and reduce the plasma FPA level. Thus, the transient TAT elevation following AT III administration may not only be due to extraction of thrombin from the fibrin clots of thrombi but also to intravascular thrombin which is not attached to thrombi. FPA is the best molecular marker for thrombin hyperactivity and it should be monitored in AT III-deficient pregnant women in the third trimester.     

Specific binding of thrombin-antithrombin III complex to hepatocytes

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37°C: the apparent Km value is 0.8 /uM. The rate of binding is approximately 1.6 × 10⁵ molecules h^?1 cell^?1 at this concentration. At 4°C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.     

Interaction of heparin with histidine-rich glycoprotein and with antithrombin III

The interaction between heparin, histidine-rich glycoprotein and antithrombin III was studied in purified systems. Histidine-rich glycoprotein binds heparin and thereby interferes with its interaction with antithrombin III, resulting in neutralization of the anticoagulant activity. This interaction occurs with clinical grade heparin as well as with high affinity (for antithrombin III) heparin and with a high affinity heparin fragment with Mr 4,300. Low affinity heparin competes with high affinity heparin for the binding to histidine-rich glycoprotein which results in an apparent increase of the anticoagulant activity of high affinity heparin. The interaction between heparin and histidine-rich glycoprotein is counteracted by Ca2+-binding anticoagulants, indicating that it is dependent on the presence of divalent metal ions. Ethylenediaminetetraacetate is a much more potent inhibitor of the interaction between heparin and histidine-rich glycoprotein than citrate.     

Elastase-like activity in cultured aortic endothelial cells

Cultured porcine aortic endothelial cells were studied for cellular and secreted elastase activity. We describe an activity hydrolyzing the synthetic elastase substrate, succinyl(alanine)3 nitroanilide, but not elastin, which was shown to be membrane located and was not secreted to the culture medium. A different neutral proteinase activity degrading insoluble elastin was demonstrated in the culture medium following its fractionation by gel filtration high performance liquid chromatography (HPLC). Since no elastinolytic activity could be directly detected in the conditioned medium, it is likely that the chromatographic separation removed an endogenous inhibitor.     

Interaction of 3H-defibrotide with cultured human umbilical vein endothelial cells

Defibrotide is a profibrinolytic and antithrombotic drug which seems to modulate endothelial cell function. In this study, a method for radioactive labeling of the drug and its interaction with cultured endothelial cells is proposed. 3H-Acetic anhydride was used to label defibrotide. Endothelial cells obtained by collagenase treatment of human umbilical cord veins were cultured in 24-welled plastic culture dishes. Binding experiments were carried out by incubating cell cultures with media containing various concentrations of labeled defibrotide. Our results showed that labeled defibrotide has a KL value of 4.2 micrograms/ml for endothelial cells. Although the presence of a specific transporter is possible, the high molecular weight of the fraction used suggests that the interaction is binding to a specific receptor.     

Glucose inhibits prostacyclin production by cultured aortic endothelial cells

A reduction in production of prostacyclin (PGI2) by the cells in the vascular wall may play a role in the pathogenesis of atherosclerosis in diabetic patients. The present study was undertaken to evaluate the effect of glucose on PGI2 production by endothelial cells in vitro. It was shown that PGI2 production by cultured bovine aortic endothelial cells was significantly reduced in the presence of a high concentration of glucose (300 mg/dl) compared with physiological concentrations of glucose (100 mg/dl). In contrast, no reduction in PGI2 production was observed in cells cultured with equimolar mannitol, suggesting that glucose itself, rather than the effect of osmolality, inhibited PGI2 production by cultured endothelial cells. In addition, a high concentration of glucose also inhibited the proliferation of cultured endothelial cells.     

Determination of human thrombin-antithrombin III complex in plasma with an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of thrombin-antithrombin III complex (TAT) in human plasma. The test system follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The antibodies bind selectively to the corresponding antigen moieties of TAT. The assay was calibrated with definite concentrations of preformed purified TAT added to TAT-poor plasma. The lower limit of sensitivity of the assay was 0.5 microgram/l. Mean coefficients of variation of 4.2% (intraassay) and 3.5% (interassay) were found for TAT concentrations between 2 and 60 micrograms/l. A reference range from 0.85 to 3.2 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In plasma samples from patients with pulmonary embolism (n = 17), TAT concentrations between 3 and 25 micrograms/l were measured. In 15 patients with deep vein thrombosis, TAT was found up to 3 to 25 micrograms/l. From these data we conclude that measurement of TAT can be a sensitive parameter for specific detection of a latent activation of the clotting pathway.     

Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.     

Release of heparan sulfate proteoglycans from cultured aortic endothelial cells by thrombin

Anticoagulant heparan sulfate proteoglycans have been shown to be released from cultured endothelial cells. The effect of thrombin on their release was investigated. Thrombin t more than one unit/ml accelerated the release of [as]sulfate-labeled glycosaminoglycans from cultured porcine aortic endothelial cells. The effect of thrombin reached a maximum after one hour of incubation, and was dependent on the enzyme concentration. 10 unit/ml of thrombin released approximately twice as much amount of J'S-glycosaminoglycans as did Hanks' balanced salt solution alone. When the active site of thrombin was blocked by either diisopropylfluorophosphate or hirudin, the enzyme effect was completely abolished. Released glycosaminoglycans were resistant to chondroitin ABC lyase digestions, but degraded by either heparitinase or nitrous acid treatments. Released ³⁵S-materials were precipitated with trichloroacetic acid and shown to be degraded into smaller molecules after alkali treatment on Sepharose CL-6B gel filtration chromatography. On the other hand, thrombin treatment of Cr-labeled cells did not cause the release of radioactivity. These results indicate that thrombin potentiates the release of heparan sulfate proteoglycans from cultured aortic endothelial cells without causing an appreciable damage to the cells. The effect of thrombin is active site-dependent and requires a relatively high enzyme concentration.     

Antithrombin III microheterogeneity in antithrombin III deficiency and in the antithrombin III abnormality, "antithrombin III Toyama"

Antithrombin III (AT III) microheterogeneity was investigated in 12 cases of congenital AT III deficiency and 2 cases of congenital AT III abnormality by isoelectric focusing (IEF) and immunofixation. In congenital AT III deficiency, IEF and immunofixation revealed AT III as 8 bands which was indistinguishable from normal control in terms of the number of bands and the isoelectric point (pI) of each band. In the proband of the congenital AT III abnormality, however, IEF and immunofixation showed AT III as 8 bands which shifted slightly but definitely to the acidic side compared to those of normal subjects. This change in pI of the abnormal AT III was considered to reflect the amino acid replacement in the polypeptide chain of the abnormal AT III molecule.     

Effects of dipyrone on prostaglandin production by human platelets and cultured bovine aortic endothelial cells

Dipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30--40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2. Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187. These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.