Plasminogen activator inhibitor-1 levels and activity decrease after intervention in patients with critical limb ischaemia.

OBJECTIVE/BACKGROUND: Patients with peripheral arterial occlusive disease (PAOD), in particular critical limb ischaemia (CLI), carry a high risk of thrombotic events. We hypothesised that patients undergoing conservative, endovascular, or open surgical treatment for CLI have increased levels of plasminogen activator inhibitor-1 (PAI-1), leading to a prothrombotic state. The objective was to determine levels of PAI-1 in patients with acute or chronic PAOD/CLI. METHODS: Thirty-two patients with a median age of 74 (49-90) years were included. Three underwent thrombolysis for acute limb-threatening ischaemia. Twenty-six patients with chronic ischaemia received endovascular (n = 20) or open (n = 6) surgical treatment. Three were treated conservatively. Biomarkers and ankle brachial index (ABI) were measured before and up to 1 month after intervention. Patency was studied with repeated duplex ultrasound. RESULTS: Ankle pressure and ABI improved after intervention (p < .001). C-reactive protein (CRP) increased from a median of 7.90 mg/L at baseline to 31.5 on day 1 (p < .001), 28.0 on day 6 (p < .001), and returned to baseline levels on day 30. PAI-1 antigen and activity decreased from day 6 and onwards post-intervention compared with baseline (p < .05). A great individual variability in PAI-1 antigen and activity was observed. Although most actively treated patients had normal PAI-1 activity, 11/29 (38%) were above that level of normality at baseline, 10/24 (42%) on day 1, 3/23 (13%) on day 6, and 5/27 (19%) on day 30 after intervention. CONCLUSION: Endovascular and open surgical treatment resulted in improved ankle pressure and ABI. The intervention was followed by a transient increase in CRP and a sustained reduction in PAI-1 levels and activity.

Safety assessment of new antithrombotic agents: lessons from the EXTEND study on ximelagatran.

BACKGROUND: Ximelagatran, the first oral direct thrombin inhibitor, was shown to be an effective antithrombotic agent but was associated with potential liver toxicity after prolonged administration. OBJECTIVES AND METHODS: The aim of the EXTEND study was to assess safety and efficacy of extended administration (35 days) of ximelagatran or enoxaparin for the prevention of venous thromboembolism after elective hip replacement and hip fracture surgery. A follow-up period, including assessment of liver enzymes (in particular alanine aminotransferase; ALAT), until post-operative day 180 was planned, with visits at days 56 and 180. RESULTS: Randomization and administration of study drugs were stopped following a report of serious liver injury occurring 3 weeks after completion of ximelagatran treatment. At the time of study termination, 1158 patients had been randomized and 641 had completed the 35-day treatment; with 303 ximelagatran and 265 enoxaparin patients remaining in the study through to the day 56 follow-up visit. Overall, 58 patients showed an ALAT increase to >2x upper limit of normal: 31 treated with enoxaparin, 27 with ximelagatran. Three ximelagatran patients also showed symptoms potentially related to liver toxicity. Eleven ximelagatran patients showed an ALAT increase after study treatment ended. The clinical development of ximelagatran was terminated and the drug withdrawn from the market. Evaluation of the relative efficacy of the two treatments as specified in the protocol was impossible due to the premature termination of the study. CONCLUSIONS: Prolonged administration of ximelagatran was associated with an increased risk of liver toxicity. In a substantial proportion of patients, ALAT increase occurred after treatment withdrawal. The findings seen with ximelagatran should be considered when designing studies with new antithrombotic agents.

Long-term treatment with ximelagatran, an oral direct thrombin inhibitor, persistently reduces the coagulation activity after a myocardial infarction.

BACKGROUND: In the ESTEEM study, patients with a recent myocardial infarction were treated with aspirin and randomized to one of four doses (24-60 mg b.i.d) of the oral direct thrombin inhibitor ximelagatran or placebo for 6 months. Ximelagatran and aspirin reduced the risk of recurrent ischemic events compared with aspirin alone. In the present substudy we evaluated the different doses of ximelagatran on pharmacokinetics as measured by plasma concentration of the active compound melagatran and activated partial thromboplastin time (APTT) and pharmacodynamics as related by markers for coagulation activity, prothrombin fragment 1 + 2 (F1 + 2) and D-dimer. METHODS AND RESULTS: Plasma samples from 518 patients were collected before, during and after the treatment period. There was a linear dose-concentration relation at peak and trough and a linear relation between concentration and APTT (P < 0.001). F1 + 2 and D-dimer were decreased by 25% and 52% at 1 week (P < 0.001) in the ximelagatran groups compared with the placebo group and the reductions were maintained during the 6 months treatment. There were no differences detected in F1 + 2 or D-dimer levels between the different ximelagatran dosages. There was no correlation between the melagatran concentration and the change in F1 + 2 and D-dimer levels. After cessation of ximelagatran F1 + 2 and D-dimer levels returned to the initial levels. CONCLUSION: The dose of ximelagatran and APTT are linearly related to the plasma concentration of melagatran. Ximelagatran induces a sustained and stable reduction of thrombin generation and fibrin turnover without any relation to dose above 24 mg b.i.d. These properties indicate that long-term treatment with a low dose of ximelagatran may provide valuable depression of coagulation activity in aspirin treated post myocardial infarction patients.

Visualization of monocyte recruitment into atherosclerotic arteries using fluorescent labelling.

It has been shown that monocytes are present in early atherosclerotic lesions and in mechanically-injured arterial intima, but direct morphological tracing of specific leukocyte populations into such areas has been lacking. A method for FITC-labelling of leukocytes was therefore evaluated for monocyte studies. Monocyte (95% pure) populations were isolated from blood by counterflow centrifugation and labelled by incubation with free fluorescein isothiocyanate 1-hydrochloride (FITC) in Hank's balanced salt solution. FITC-labelled monocytes showed glass adherence, spreading and migration, as well as acid phosphatase positivity and phagocytosis for up to 20 days in tissue culture. For in vivo experiments, hypercholesterolemic (H) and normal (N) swine were bled repeatedly, and monocyte populations were isolated, labelled and reinjected. Labelled cells were found in blood samples. Animals were killed after 9 days, and formaldehyde-fixed and frozen samples of aortae were studied en face and/or sectioned and examined microscopically under fluorescence. FITC-labelled leukocytes could be found adherent to sites of thickened intima but not to normal areas. Labelled cells were also detected within atherosclerotic lesions. These results show the feasibility of the labelling technique and provide direct visualization of monocyte recruitment from the blood into atherosclerotic and lesion-prone areas.

Effect of venous stasis on vessel wall fibrinolysis.

The effects of prolonged venous stasis on release of plasminogen activators (PA) from the caval vein of rats were studied. PA activity was continuously reduced in the intimal layer and after 8 hr of stasis the reduction was statistically significant. After 2 and 3 days no intimal PA activity was found. The PA activity in the adventitial vasa vasorum was unchanged during the experimental period.

Factor V Leiden (G1691A) and prothrombin gene G20210A mutations as potential risk factors for venous thromboembolism after total hip or total knee replacement surgery.

Patients (n = 1600) from 12 European countries, scheduled for elective orthopaedic hip or knee surgery, were screened for Factor V Leiden and prothrombin gene G20210A mutations, found in 5.5% and 2.9% of the populations, respectively. All patients underwent prophylactic treatment with one of four doses of melagatran and ximelagatran or dalteparin, starting pre-operatively. Bilateral ascending venography was performed on study day 8-11. The patients were subsequently treated according to local routines and followed for 4-6 weeks postoperatively. The composite endpoint of screened deep vein thrombosis (DVT) and symptomatic pulmonary embolism (PE) during prophylaxis did not differ significantly between patients with or without these mutations. Symptomatic venous thromboembolism (VTE) during prophylaxis and follow-up (1.9%) was significantly over-represented among patients with the prothrombin gene G20210A mutation (p = 0.0002). A tendency towards increased risk of VTE was found with the Factor V Leiden mutation (p = 0.09). PE were few, but significantly over-represented in both the Factor V Leiden and prothrombin gene G20210A mutated patients (p = 0.03 and p = 0.05, respectively). However, since 90% of the patients with these genetic risk factors will not suffer a VTE event, a general pre-operative genotyping is, in our opinion, of questionable value.

Prevention of postoperative pericardial adhesions by closure of the pericardium with absorbable polymer patches. An experimental study.

Pericardial adhesions after cardiac operations are a widely known phenomenon. They may severely complicate reoperations, making reentry hazardous, increasing bleeding, and prolonging the operation time. The anatomic orientation and visibility of both bypass grafts and coronary arteries are also impaired. With the aim of minimizing pericardial adhesions after cardiac operations, we studied the course of tissue regeneration after implantation of a new absorbable patch made from poly-hydroxy-butyrate. A total of 23 sheep were studied. Of these, 18 formed the test group and five served as control animals. The animals were killed at intervals of 2 to 30 months after the operation. In 14 of the 18 test animals no adhesions developed. In three animals loose adhesions were found, and in one with signs of postoperative infection there were moderate, generalized adhesions. All control sheep showed moderate adhesions; no infection was noted in this group. Light microscopy in the test group revealed a layer of mesothelium-like cells facing the epicardial side; this was already present in the early specimens. Poly-hydroxy-butyrate appeared to be slowly phagocytosed by polynucleated macrophages, which were still found occasionally in the late samples. Lymphocytes and platelets were rare. Scanning electron microscopy showed, on the epicardial side of the regenerated tissue, a mesothelium-like lining that completely covered the underlying collagen layer. The surface cell morphology grossly resembled that of native pericardium. It was concluded that in this animal model poly-hydroxy-butyrate pericardial patches decreased adhesions and preserved coronary anatomy. The findings in the control group demonstrated that pericardial surgery in the sheep was associated with adhesion formation.

Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results.

Autologous endothelial seeding of thin-walled ePTFE vascular prostheses (I.D.4 mm), interposed in the carotid artery, was performed in 10 dogs and 14 sheeps. Aspirin (250 mg/day) and dipyridamole (75 mg/twice daily) were given throughout the study as antiplatelet therapy. Animals were killed 2 and 5 weeks after surgery. Patency rates for seeded grafts in dogs were 75% (6:8) and 83% (10:12) at 2 and 5 weeks, respectively. In control grafts the patency rates were identical. Patency rates for seeded grafts in sheep were 0% (0:5) and 11% (1:9) at 2 and 5 weeks, respectively. Control grafts in sheep had a patency rate of 40% (2:5) and 0% (0:9) at corresponding times. Scanning electron microscopy showed an almost complete endothelialization of seeded grafts in dogs after 5 weeks. Platelet deposition was studied in the dogs by means of chromium-51-labeled autologous platelets. Significantly fewer platelets accumulated on seeded grafts, and the ratio of 6-keto-PCF1 alpha to thromboxane B2 was significantly higher, compared with unseeded grafts, which indicated the presence of a functionally active endothelial lining in seeded grafts. Differences in the hemostatic system could account for the high clotting incidence in sheep, compared with that in dogs. Such species differences make extrapolations to the clinical situation from autologous endothelial seeding in experimental animals hazardous.

Heparin coating reduces blood cell adhesion to arterial filters during coronary bypass: a clinical study.

Deleterious effects of cardiopulmonary bypass (CPB) are partly sequelae of blood-foreign surface reactions. Coating the inner surfaces of CPB sets with heparin has been shown to decrease activation of humoral cascade systems. The aim of this study was to investigate whether the heparin-coated CPB sets influence adhesion of blood cells to surfaces of arterial filters during CPB. Thirty-one patients undergoing coronary artery bypass grafting were studied. In the control group (C) standard CPB sets and standard doses of heparin (300 IU/kg) were employed; in the HC (heparin-coated) group, heparin-coated CPB sets and reduced heparin doses (range, 150 to 225 IU/kg) were used. Two additional groups were also studied; group FC (coated filter), with standard CPB sets and heparin-coated arterial filters (heparin dose, 300 IU/kg), and group OC (uncoated filter), with heparin-coated CPB sets and standard arterial filters (heparin dose, 300 IU/kg). The inner surfaces of the arterial filters were examined after CPB with scanning electron microscopy. Scanning electron microscopy demonstrated almost clean surfaces in heparin-coated filters even when other parts of the circuit were uncoated. Using an arbitrary adhesion score, significant differences between the groups were noted in the adhesion grade; it was lowest in group HC (2.2 +/- 0.27 [mean +/- standard error of the mean]) versus group C (5.4 +/- 0.53; p < 0.001). In group FC it was marginally higher than in group HC but almost significantly lower than in group OC (2.6 +/- 0.68 versus 5.4 +/- 0.81; p = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular trauma induced by clamping- correlation between surface ultrastructure and fibrinolytic activity.

The effects of application of a vascular clamp for 30 sec were studied on the femoral vessels in rats. Control and injured vessel segments were studied with scanning electron microscopy and with a histochemical method to estimate the fibrinolytic activity in the vessel walls. Endothelial injury with loss of endothelial cells was induced by application of the clamp. This was correlated to a reduction of the fibrinolytic activity in the vessel wall. In arteries re-endothelialization was seen three weeks after induction of the trauma, and it was completed after eight weeks. Venous lesions healed earlier. The re-endothelialization was followed by a return to normal fibrinolytic activity.

Scanning electron microscopy - some methodological considerations.

Although scanning electron microscopy has been used in arterial research for more than a decade, methodological standards are still subject to dispute. In this presentation, our experience with different methods to prepare arterial tissue is described. Our efforts have been aiming at optimizing the reproducibility of our preparations. The recommended procedure involves fixation at "physiological" arterial pressure in Karnovsky's fixative for a short period, post-fixation by immersion, drying of the tissue with the critical point method and covering it with gold in a sputtering device. The effects of various deviations from this procedure are described. After preparation with the recommended procedure a resolution of 200-400 A has been obtained. General characteristics of the arterial endothelium are concurrent with those described in studies using light microscopy or transmission electron microscopy.

Early reactions of the arterial wall, following mechanical trauma. A scanning and transmission electron microscopy study.

Endothelial injury has for a long time been suggested to be an important factor for the development of atherosclerotic lesions. In this study, a superficial, circumferential, injury to the aortic intima was induced via a catheter covered with small stones, introduced into the femoral artery of rabbits. The immediate answer of the arterial wall and the formed elements of the blood was studied with combined scanning and transmission electron microscopy. De-endothelialization was noted and the denuded areas were almost immediately covered by platelets, spreading over the surface and releasing the contents of their granules by time. At some locations deeper injuries to the elastic laminae of the wall could be noted. Especially frequent around these areas, but also common all over the de-endothelialized surface, leukocytes were seen. Both neutrophils, lymphocytes and monocytes could be found. A demarcation of the border of the injury was noted three days after catheterization.

Endothelial surface ultrastructure following hypoxia and hyperoxia.

Effects of hypoxia and hyperoxia on the ultrastructure of aortic endothelial cells were studied in rats. Rats were subjected to FIO2 0.05, 0.06 or 0.1 for various period times. Hypoxia was followed by nuclei protrusion, appearance of holes through the luminal membranes of individual cells. Some cells showed signs of partial of total disintegration. Hyperoxia (FIO2 = 1.0) were followed by slight morphological changes notable after 8 hours but most pronounced after 24 hours. The present study corroborated previous studies in that both hypoxia and hyperoxia can alter endothelial integrity.

Endothelial fibrinolysis and ultrastructure following graded mechanical trauma.

For study of the progression and resolution of endothelial damage following mechanical vascular trauma in rats, the effects of a clamp with hydrostatically controlled occlusion pressure were compared with those of a standard mechanical clamp. The endothelial morphology was investigated with scanning electron microscopy and its function with histochemical analysis of plasminogen activator activity. Arterial or venous compression with hydrostatic pressure 1.5 kPa (11 mmHg) for 5 or 20 min produced de-endothelialized areas in veins, but only slight endothelial elevation in arteries. Hydrostatic occlusion of arteries with 12 kPa (90 mmHg) for 30 sec did not affect endothelial morphology and fibrinolytic activity, but after occlusion for 5 or 20 min the endothelium in both arteries and veins was severely damaged. The effects of hydrostatic (12 kPa) clamping on endothelial morphology and function after 5 or 20 min did not differ from those of mechanical (48 kPa = 360 mmHg) clamping. Apposition of vascular walls, except for the shortest time (30 sec) thus injured the endothelium, but minimizing occlusion pressure may reduce structural damage in the vessel wall.

Local fibrinolytic activity and its structural basis in experimental gastric ulcers.

The stomach contains plasminogen activators localized to submucosal vessels and to the mucosa. To study the release of plasminogen activators from mucosal cells, we induced mucosal damage by pyloric ligature and instillation of 0.5 mol/l HCl in rats. After 4 h, all rats developed focal necrotic lesions and a diffuse mucosal damage, as studied both by light microscopy and by scanning electron microscopy. Diffuse mucosal damage was accompanied by significant increase (P less than 0.001) in mucosal fibrinolytic activity based on plasminogen activators. This activity was probably derived from injured mucosal cells since focal necrotic lesions (erosions and ulcers) with total denudation of the mucosal epithelium lacked fibrinolytic activity. The results obtained in this study support the hypothesis that plasminogen activators are released during cellular degeneration.

The effect of balloon catheter trauma on surface ultrastructure and fibrinolytic activity in the caval vein in rats.

Surgical procedures in veins such as thrombectomy are followed by a high complication rate. Re-occlusion is not uncommon in spite of primarily successful thrombectomy. The venous endothelium is damaged by the thrombotic process and the mechanical trauma from the balloon will be additive. The endothelial cells are the source of fibrinolytic activators. The present study compared changes in fibrinolytic activity in the vessel walls from the caval veins in rats to ultrastructural changes as seen in the scanning electron microscope. The balloon caused an almost complete denudation of the intima and the lesions were fully covered with platelets 1 hour after trauma. After 3 days reendothelialization had started. The fibrinolytic activity was significantly decreased at 1 and 24 hours following trauma. Reendothelialization was accompanied by return to normal fibrinolytic activity.