Behavior of plasminogen at the luminal surface of the normal and deendothelialized rabbit aorta in vivo and in vitro

The behavior of purified rabbit plasminogen at the luminal surface of the uninjured and deendothelialized rabbit aorta has been studied in vivo and in vitro. After intravenous injection, 125I-plasminogen associated rapidly with the endothelium (approximately 0.1 pmol/cm2 at saturation) and passed through to accumulate in the subendothelium. At two to 15 hours after injection, 11 to 15 times more radioactivity was associated with the subendothelium than with the endothelium. Removal of the endothelium by balloon catheter led to a rapid adsorption of 125I-plasminogen by the luminal surface of the vessel; saturation (9.1 pmol/cm2) was attained at ten to 20 minutes after deendothelialization. Of the adsorbed plasminogen (radioactivity), only 2% to 4% was associated with the adherent platelet monolayer. Uptake of 125I- plasminogen by the deendothelialized vessel was not significantly inhibited by epsilon-aminohexanoic acid whether injected before or after the 125I-plasminogen. No evidence of plasmin activity at the aorta surface was found from either transmission electron microscopy studies or from amidolytic assays of plasminogen-saturated deendothelialized aorta samples before or after urokinase treatment. Balloon catheter treatment in vivo, however, generated significant antiplasmin activity of the deendothelialized aorta surface. We conclude that plasmin formed in vivo is probably inactivated by the antiplasmin activity that is associated with the subendothelium.     

Metabolism-permeability coupling in the normal rabbit aorta

Microfluorimetric and quantitative cytoenzymatic techniques were employed to examine regional variations in endothelial macromolecular uptake as related to inner mural intermediary metabolism in aortas from normocholesterolemic rabbits. Concomitant reductions in luminal fluorescein isothiocyanate-conjugated bovine serum albumin (FITCBSA) accumulation and succinate (SDH), lactate (LDH) and glucose-6-phosphate (G-6-PDH) dehydrogenase activities were evidenced from ascending to upper abdominal segments. Further diminution of FITCBSA accumulation was observed in the lower abdominal aorta, whereas there was a corresponding elevation of enzyme activities. Highly significant but not exceedingly close correlations were obtained between luminal FITCBSA uptake and inner mural SDH and LDH activities. However, much of the associated variability was attributable to the lower abdominal segment, where there was a microscopically-discernible augmentation in adventitial surface FITCBSA accumulation. The overall data provide direct in vivo support for the concept that endothelial macromolecular transport is coupled to mural oxidative demands, but also indicate that luminal metabolism-permeability relationships are influenced by factors such as extensiveness of the vasoral network and wall thickness. Details of the metabolism-permeability coupling hypothesis and observations implicating metabolic events as basic causative factors underlying vascular pathogenesis are discussed.     

The interaction of convection and diffusion in the transport of 131I-albumin within the media of the rabbit thoracic aorta

The interaction of convection and diffusion in the transport of 131I-labeled albumin within the wall of rabbit thoracic aorta was studied in vessels excised at in vivo length. They were pressurized with a solution containing no tracer and immersed in a solution containing labeled albumin. The label then entered the wall tissue via the adventitia and had to diffuse against the convective flux which occurred from the lumen to the adventitia. Experiments were performed on intact and deendothelialized vessels pressurized to 70 and 180 mm Hg. At the end of each experiment the vessels were subjected to sequential frozen sectioning parallel to the lumenal surface. The radioactivity of the 20-micron-thick sections was determined and expressed as a tissue:labeled solution concentration ratio. Transmural profiles of these ratios were thus obtained. The steady state was found to be achieved by about 90 minutes. When the convection was enhanced by removal of the endothelium, the average ratios were lower than when the endothelium was intact, and the profile was much flatter. The results suggest that convection influenced macromolecular transport within the arterial wall, even in vessels with intact endothelium.     

Evidence that rabbit 125I-antithrombin III binds to proteoheparan sulphate at the subendothelium of the rabbit aorta in vitro

The endothelium of the rabbit thoracic aorta was removed from the vessel wall by one of two procedures, and the freshly exposed subendothelial surface was used for 125I-antithrombin III binding studies. Pretreatment of the subendothelium with either heparitinase or thrombin diminished the uptake of 125I-antithrombin III by up to 80%, whereas pretreatment with plasmin, hyaluronidase or FPR thrombin had little effect. Morphometric analysis of the subendothelium from enzyme-treated and -untreated tissues showed that, whereas plasmin, thrombin and heparitinase each caused a dramatic reduction of the large proteoglycan granules of the extracellular matrix, only exposure to heparitinase and thrombin caused a reduction in the small proteoglycans which populate the basement membrane of smooth muscle cells. Of the subendothelium-bound 125I-antithrombin III, more than 80% was efficiently removed by excess thrombin or by excess heparin. Evidence was obtained for the formation of high molecular weight thrombin-antithrombin III complexes. We conclude that antithrombin III binds largely to proteoheparan sulphate located in the basement membrane of the intimal smooth muscle cells for the purpose of inactivating certain proteases which arise during haemostatic change.     

Role of LDL receptors in the in vitro uptake and degradation of LDL in the media of rabbit thoracic aorta

The possible role of plasma low-density protein (LDL) receptors in the uptake and degradation of LDL in the whole arterial wall was investigated by comparison of the in vitro uptake of 125I-native LDL (nLDL) and 131I-methylated LDL (mLDL) by the media of deendothelialized rabbit thoracic aorta excised at in vivo length and pressurized to 70 mm Hg, taking the advantage that mLDL is not recognized by the LDL receptor. The distribution of the relative concentrations of nLDL (Cn) and mLDL (Cm) across the wall was obtained using a serial frozen sectioning technique. The aorta was incubated under three different conditions for varying periods of incubation in order to analyze separately the processes of binding, binding-internalization, and degradation. At 39 degrees C, in which binding-internalization and degradation occurred, Cn was significantly higher than Cm at each position across the media. The mean medial Cn/Cm ratio was 1.36 +/- 0.15 (n = 5) after 1 hour of incubation, and decreased to 1.23 +/- 0.22 (n = 7) after 2 hours of incubation and to 1.13 +/- 0.11 (n = 5) after 4 hours of incubation. At 4 degrees C, in which internalization and degradation were blocked, the Cn/Cm ratio reflected the surface nLDL binding alone; the Cn/Cm ratio was 1.47 +/- 0.20 (n = 5) after 4 hours of incubation, higher than the value obtained at 39 degrees C. To investigate whether degradation of nLDL occurred after receptor binding, the interstitial LDL was washed out by an LDL-free solution after 2-hour incubation at 39 degrees C. After 30 minutes of washout, the Cn/Cm ratio decreased to 1.06 +/- 0.20 (n = 5) in the inner media and was unchanged in the outer media. After 1 hour of washout, the ratio declined to 0.57 +/- 0.18 (n = 7) in the inner part of the media and increased progressively to 1 at the media-adventitia boundary. The Cn/Cm ratio, at 0.67 +/- 0.12 (n = 5), was practically constant throughout the media after 2 hours of washout. The nLDL degradation rate across the media was obtained from the comparison of nLDL and mLDL before and after the washout. A steep decreasing gradient in nLDL degradation rate was observed from the luminal to the external surface. The mean medial nLDL degradation rate value was 9.6 +/- 4.5 microliters/cm3 wet tissue/hr. We concluded that functional LDL receptors participate in the uptake and degradation of LDL in the whole aorta.     

O-methylation of isoproterenol by the endothelium of the rabbit thoracic aorta

We have examined the influence of endothelial cell removal upon the O-methylation of the isomers of isoproterenol in the isolated rabbit aorta. Endothelial cells were removed by mechanically abrading isolated segments of rabbit aorta. The latter procedure resulted in the abolition of acetylcholine-mediated relaxation of tissues, a process dependent upon the presence of endothelial cells. In addition, histological staining and electron-microscopic analysis indicated the presence of endothelial cells in tissues not subjected to mechanical abrasion and the absence of endothelial cells in tissues subjected to abrasion. Moreover, ultrastructural analysis revealed that the influence of abrasion was limited to the luminal side of the internal elastic lamina. The removal of endothelial cells from the isolated rabbit aorta was associated with a decrease (approximately 30%) in the O-methylation of the isomers of isoproterenol. Inhibition of the extraneuronal uptake process with deoxycorticosterone (DOCA) produced a predicted decrease in the O-methylation of isoproterenol in tissues with the endothelium intact. In the absence of endothelial cells, the inhibition of the O-methylation of isoproterenol mediated by DOCA persisted. The results suggest that the O-methylation of catecholamines can occur in both medial and endothelial structures in this blood vessel and support the results of immunohistochemical studies that suggested a presence of catechol-O-methyltransferase in vascular endothelial structures. In addition, the findings exclude a possibility that the extraneuronal uptake process for catecholamines resides exclusively in endothelial structures. The functional consequences of vascular endothelial cell O-methylation are discussed.     

Treatment of venous thrombosis in antithrombin III deficient patients with concentrates of antithrombin III

Summary Two patients with familial antithrombin III deficiency developed deep venous thrombosis of the lower limb. The diagnosis of venous thrombosis was made by the indium labelled platelet technique which also allowed for the daily assessment of thrombus size. Each patient received treatment with Warfarin, subcutaneous heparin, and infusions of antithrombin III concentrates. The authors conclude that infusions of antithrombin III concentrates may be of value in limiting the extent of acute thrombosis in patients with a severe deficiency of this protein and may help prevent pulmonary embolism. The haemorrhagic risk of continuing modest doses of heparin with high dose ATIII therapy appears small. In addition to its value in the diagnosis of venous thrombosis the indium platelet technique may give an early indication of thrombus extension and may thus indicate the effectiveness of treatment.     

Treatment of venous thrombosis in antithrombin III deficient patients with concentrates of antithrombin III

Summary Two patients with familial antithrombin III deficiency developed deep venous thrombosis of the lower limb. The diagnosis of venous thrombosis was made by the indium labelled platelet technique which also allowed for the daily assessment of thrombus size. Each patient received treatment with Warfarin, subcutaneous heparin, and infusions of antithrombin III concentrates. The authors conclude that infusions of antithrombin III concentrates may be of value in limiting the extent of acute thrombosis in patients with a severe deficiency of this protein and may help prevent pulmonary embolism. The haemorrhagic risk of continuing modest doses of heparin with high dose ATIII therapy appears small. In addition to its value in the diagnosis of venous thrombosis the indium platelet technique may give an early indication of thrombus extension and may thus indicate the effectiveness of treatment.     

Thrombosis of an apparently normal thoracic aorta and arterial embolism

With the advent of new imaging techniques, the aorta has been increasingly identified as a source of arterial embolism. The majority of thrombi occur in aneurysms or are adherent to atherosclerotic lesions in the abdominal aorta. Thrombi in the thoracic aorta are much less common, particularly in apparently normal aortas. Consequently, the natural history and optimal treatment of these lesions are not well-defined. The aim of this article was to describe the clinical characteristics, treatment, and outcome in three patients with thoracic aorta thrombosis and arterial embolism. Currently available literature on this pathology is reviewed and the differential diagnosis of these lesions is discussed.     

Further investigations on antithrombin III in the plasmas of patients with the abnormality of antithrombin III Budapest.

Antithrombin III (AT-III) was studied in a thrombophilic family with an abnormal AT-III molecule (antithrombin III Budapest) using a modified crossed immunoelectrophoresis technique, gel filtration, 'rocket' immunoelectrophoresis and a heparin cofactor assay. When agarose was applied in the first phase of the crossed immunoelectrophoresis, the normal and the pathological AT-III revealed identical electrophoretic mobility. However, when heparin was mixed with agarose in the first phase of electrophoresis, the propositus' plasma displayed a different AT-III pattern from normal plasma. His plasma contained the first component of the normal plasma (Immune Antithrombin III1, IAT-III) in a concentration of only 5% of normal, and a protein in high concentration which although immunoreactive to AT-III antisera, had an electrophoretic mobility similar (but not identical to that of IAT-III2. This abnormal protein had no heparin cofactor activity and a molecular size greater than normal plasma AT-III. Unlike AT-III, the addition of heparin did not change the molecular size of the pathogic AT-III molecule significantly. The abnormal protein was present in lower concentrations in the patient's children and at the time of study they had no clinical or laboratory evidence of intravascular coagulation.     

Relevance of antithrombin III in thrombogenesis: the diagnosis and therapy of antithrombin III defects

Pathological and inconspicuous AT III measuring values were compared with the clinical findings (arterial occlusive diseases, postthrombotic syndromes, acute profound thrombosis of the pelvic veins and the leg veins, acute heparin tolerance in several basic diseases). After calculatory elaboration of all rightly positive and falsely positive, rightly negative and falsely negative laboratory results becomes evident that the positive power of prognosis of the AT III determination is unsatisfactory for a certain basic disease and the negative evidence in these questionings rather well satisfy for the functional measuring method. - The sensitivity of the functional AT III test for the recognition of increased heparin tolerances is quite satisfactory, and the test for the answer of this questioning also suitable and more sensitive than the immunological proof method. - The high percentage of rightly positive and rightly negative findings of all tested results of apparently healthy persons and the groups of patients represented makes clear, which role plays the AT III as a physiologic inhibitor against coagulation-active serine proteases particularly in DIC and in profound thrombosis of the pelvic and leg veins. - For the observation of the course in profound thrombosis of the pelvic and led veins, features of postthrombotic condition and the DIC at least the functional determination of AT III is decisively important.     

一氧化氮在高脂饲养兔胸主动脉的变化和意义

观察高脂饲养兔胸主动脉血管环对去甲肾上腺素（ＮＥ）的反应性及环磷酸鸟苷（ｃＧＭＰ）含量的变化。结果表明其对ＮＥ的反应性降低，组织ｃＧＭＰ的含量减少。左旋精氨酸（Ｌ－Ａｒｇ）和硝普钠（ＳＮＰ）可恢复此反应性，而一氧化氮合成酶（ＮＯＳ）阻滞剂Ｌ－硝基精氨酸（Ｌ－ＮＮＡ）却加重其降低的反应性。提示高脂饲养兔血管环中的一氧化氮（ＮＯ）减少，Ｌ－Ａｒｇ及ＳＮＰ可以逆转之，暗示Ｌ－Ａｒｇ／ＮＯ通路障碍可能是动脉粥样硬化的又一机理。     

Comparative effects of atrial polypeptide and neurohormone C on the interaction of factor Xa with antithrombin III

The effects of atrial polypeptide and neurohormone C upon the interaction of human factor Xa (FXa) and human antithrombin III (ATIII) were followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pattern of bands consisting of a 1 degree duplex complex (FXaalpha-ATIII band of 109 kDa, FXabeta-ATIII band of 104 kDa), a 2 degree duplex complex (alpha band of 99 kDa, beta band of 95 kDa), a 3 degree duplex complex (alpha band of 66 kDa, beta band of 62 kDa), modified ATIII (ATIIIM, 58 kDa), native ATIII (55 kDa), FXaalpha (52 kDa), FXabeta (47 kDa), and a FXa degradation product (FXagamma, 35 kDa) was detected and quantitated. Preincubation of FXa, ATIII, or mixtures thereof with atrial polypeptide produced a shift from FXaalpha-ATIII to FXabeta-ATIII complexes and increases in both ATIIIM and FXagamma, reflecting degradation of the 1 degree and 2 degree complex to form the 3 degree complex. Atrial polypeptide appeared to promote FXa-ATIII complex formation when preincubated with ATIII, or when added within 1 min to FXa/ATIII mixtures. However, when atrial polypeptide was preincubated with FXa, inhibition of the 1 degree complex formation was suggested. Upon incubation of FXa, ATIII, or mixtures thereof with neurohormone C, there was an increase in total complex formation, a decrease in ATIIIM, a decrease in FXagamma, and little change in the ratio of free FXaalpha to FXabeta, or the ratio of FXaalpha-ATIII to FXabeta-ATIII complexes. Therefore, neurohormone C may act to suppress hydrolysis or proteolytic actions of excess FXa on FXa-ATIII complexes, or autolytic activity of FXa, to the level of FXagamma via an, as yet, unknown mechanism. Additionally, neurohormone C retards the hydrolysis of the FXa-ATIII complexes which form free FXa and ATIIIM. Hence, the role of atrial polypeptide in mixtures with ATIII and in mixtures with FXa is quite contrasting, and may reflect mechanistic effects of the atrial polypeptide molecule, as well as tissue-specific reactions.     

Sympatholytic properties of several AT1-receptor antagonists in the isolated rabbit thoracic aorta

OBJECTIVE: To evaluate the facilitating effect of angiotensin II on sympathetic neurotransmission to quantitatively compare the sympatho-inhibitory potencies of the selective AT1 -receptor antagonists losartan, irbesartan and telmisartan in the isolated rabbit thoracic aorta. DESIGN: To investigate the influence of pharmacological compounds on pre-junctional sympathetic transmission, the quantification of sympathetic transmitter release is the most straightforward approach. METHODS: To investigate the sympatholytic properties of AT1 -blockers, we studied their effects on the enhancement by angiotensin II of electrical field stimulation (EFS)-evoked (2 Hz) sympathetic transmission in a modified spillover model. RESULTS: Angiotensin II (0.01 nmol/l-0.1 micromol/l) caused a concentration-dependent enhancement of EFS-evoked noradrenaline release (control versus concentrations 0.1 nmol/l-0.1 micromol/l, P<0.05). The maximal augmentation, by almost 100%, was observed at a concentration of 1 nmol/l (FR2/FR1, 2.03 +/- 0.11 versus control, 0.99 +/- 0.03). Higher concentrations (up to 0.1 micromol/l) produced less than maximal facilitation. The AT1 -receptor antagonists losartan (0.1 nmol/l-0.1 micromol/l), telmisartan (0.01-10 nmol/l) and irbesartan (0.1 nmol/l-0.1 micromol/l) concentration dependently attenuated the angiotensin II-mediated (1 nmol/l) enhancement of EFS-evoked sympathetic outflow. The concentrations that reduced the enhancement by 50% (IC50 values, expressed as -log mol/l +/- SEM) were 9.05 +/- 0.16 losartan, 10.28 +/- 0.20 telmisartan and 9.20 +/- 0.23 irbesartan. Accordingly, the order of potency with respect to sympatho-inhibition proved telmisartan> irbesartan = losartan (where > signifies P<0.05). CONCLUSIONS: The facilitating effect of angiotensin II on the sequelae of neuronal stimulation appears to be mediated by pre-synaptically located AT1 -receptors. Facilitation can be concentration dependently attenuated by AT1 -blockade. The order of potency with respect to sympatho-inhibition is telmisartan irbesartan = losartan. These differences may be explained by differences in affinity for the pre-synaptic AT1 -receptor.     

Pharmacokinetic and antithrombotic properties of two pentasaccharides with high affinity to antithrombin III in the rabbit: comparison with CY216

This study compares the pharmacokinetic and the antithrombotic properties of two pentasaccharides with high affinity to antithrombin III with those of a conventional low molecular weight heparin, CY216, in the rabbit. On a weight basis, SR 90107A/ORG 31540 (natural pentasaccharide [NPS]) and SR 80027A/ORG 31550 (sulfated pentasaccharide [SPS]) were, respectively, 4.7 and 26 times more potent antifactor Xa inhibitory agents than CY216. They were devoid of antithrombin activity, whereas the antifactor Xa/antithrombin ratio of CY216 was 3.8. After bolus intravenous administration, the clearance (mL/kg/h) of CY216 decreased from 91 +/- 27 for the dose of 12.5 U/kg to 49 +/- 14 for the dose of 50 U/kg and then remained constant up to the highest dose tested (500 U/kg). The clearance of NPS was unrelated to the dose and comparable to that of CY216 over 50 U/kg, whereas that of SPS was 10 times lower. Consistent results were observed after continuous intravenous infusions for 9 hours and subcutaneous administration. The duration of the antithrombotic effect was compared after a single subcutaneous injection of 250 U/kg of either compound in the stasis-Wessler model using human serum as thrombogenic stimulus. Two hours after the injection, the three compounds provided a thrombus prevention of greater than 95% and mean plasma activities of 0.8, 0.9, and 1.9 U/mL for CY216, NPS, and SPS, respectively. Twelve hours after injection, the antithrombotic effects of CY216 and NPS had totally vanished, whereas that of SPS was 68%. At that time, the plasma anti-Xa activities were less than 0.06 U/mL for CY216 and NPS, but 1.1 U/mL for SPS. For the latter compound, significant antithrombotic effects and detectable anti-Xa activities were still recorded 48 hours after the injection. The antithrombotic potency of the three compounds was also compared as their ability to inhibit the growth of a standardized venous thrombosis during 4 hours. The lowest total doses providing the maximum inhibitory effect were 3,125, 1,428, and 62 micrograms/kg for CY216, NPS, and SPS, respectively. These doses generated mean steady state antifactor Xa activities of 1.06, 1.5, and 1.2 anti-Xa U/mL, respectively. These observations indicate that the amplification mechanisms triggered by thrombin bound to fibrin and leading to the generation of new thrombin are essential to ensure venous thrombosis growth and that these mechanisms may be efficiently inhibited by pure antifactor Xa targeting agents.