Cloning and characterization of a muscle isoform of a Na,K-ATPase alpha subunit (SNaK1) from Schistosoma mansoni.

A cDNA encoding a Na,K-ATPase alpha subunit homologue, designated SNaK1, was isolated from an adult cDNA library of Schistosoma mansoni. The 3.8 kb DNA contained a 3021 bp open reading frame potentially encoding a 1,007 amino acid protein that had an Mr of 111,817 and a pl of 5.48. Homology searches for SNaK1 revealed approximately 70% sequence identity with a variety of Na, K-ATPases from evolutionarily diverse organisms. SNaK1 is predicted to contain 10 transmembrane regions typical of this protein family as well as other conserved domains, such as the phosphorylation site and ATP binding domain. Antibodies raised against an amino terminal peptide detected the protein in membrane preparations of eggs, cercariae and adult males and females, suggesting a general role for SNaK1. The mobility of the protein differed in various life-stages suggestive of post-transcriptional or post-translational modification. Immunolocalization of SNaK1 in sections of adult worms using epifluorescence and electron microscopy, revealed antibody labelling in the subtegumental and peripheral layers. Strong staining was discernible in the peripheral muscle band indicating that SNaK1 plays a central role in muscle contraction in adult parasites and may be the primary target of ouabain action. Staining was also detected in the secretory bodies in sections of ducts in this region and over the RER of the presumed gastrodermis. Immunogold labelling was also localized over neuronal vesicles in axons associated with the peripheral muscle layer.

Does ultrasound influence experimentally induced thrombus formation in the central artery of the rabbit ear?

BACKGROUND: Thrombosis is one of the most important causes of morbidity in the medical field. Several independent in vitro studies have shown that the fibrinolytic process may be enhanced by ultrasound, but the effect of ultrasound on thrombus formation in vivo is unexplored. The present study was designed to investigate this matter. METHODS: In a blind randomized study, standardized arteriotomies and intimectomies were performed on the central arteries of the ears of 25 rabbits. The rabbits were allocated to two groups, an untreated control group and a group treated with ultrasound (10 pulses of frequency 1 MHz and intensity 1 W/cm(2) per millisecond giving an averaged intensity of 0.01 W/cm(2)). Immediately after reperfusion, patency was confirmed by a manual empty/refill test, after which blood-flow was monitored using ultrasonic flow-probes twice a minute for two hours. At two hours, patency was rechecked. RESULTS: All vessels were patent at reperfusion, but only seven vessels (three control, four treated) were patent when flow-rate measurements started. At 2 h, patency-frequencies were 12/23 in the control group and 11/22 in the treated group. Flow-rate curves in patent vessels in both groups were similar. Microscopic investigation at one week showed no difference in thrombus accumulation. CONCLUSIONS: Ultrasound with the above characteristics does not significantly improve patency in vivo.

Neuromusculature of the ovijector of ascaris suum (Ascaroidea, nematoda): an ultrastructural and immunocytochemical study.

This study used electron microscopy and confocal scanning laser microscopy interfaced with cytochemistry to study neuromuscular interrelationships in the ovijector of Ascaris suum. An extensive nerve plexus with both FaRPergic and non-FaRPergic components extends over the outer surface of the ovijector. The non-FaRPergic component is derived from nerve branches of the ventral nerve cord, whereas the FaRPergic component emanates from two large FMRFamide-immunoreactive neurons. In the vagina vera, most myofibrils are circular in orientation and a number of them divide and run for short distances in longitudinal and diagonal directions, their myofilaments are also orientated in a variety of directions. Parallel nerve fibres run in tracts along the length of the vagina vera with branches that penetrate the muscle layers. The vagina uteri possesses a thicker hypodermis than that of the vagina vera. It appears rich in secretory and phagocytic vesicles and the luminal side is invested with an electron-dense substance. The musculature of the vagina uteri is less well developed than that of the vagina vera, being restricted to circular myofibrils, with an apparent diagonal arrangement of myofilaments. Also, the innervation is less extensive in the vagina uteri with many fibres returning to the vagina vera to rejoin the nerve net and others continuing into the uteri.

Antithrombotic and platelet activating effects of heparin in prevention of microarterial thrombosis.

It has been proposed that the limited effectiveness of heparin in preventing arterial thrombosis may be caused by heparin-mediated platelet activation. The effect of heparin on platelets and thrombus formation in vivo was investigated using a microarterial model of thrombosis involving registration of labeled platelets. Three groups of six rabbits each were treated with either saline, standard heparin, or heparin with low affinity for antithrombin III (LA-heparin), which exhibits a low anticoagulant potential but has a molecular weight distribution similar to standard heparin. (Fifty to 70 percent of standard heparin consists of low-affinity material). The heparins were infused intravenously at doses of 1 mg/kg body weight, followed by maintenance injections. Platelet accumulation was significantly increased in the LA-heparin group compared with the saline group, demonstrating heparin-induced platelet activation in vivo. In contrast to a powerful anticoagulant response observed in rabbit plasma, the antithrombotic effect of standard heparin was expressed incompletely. (Only two of three of the treated vessels remained patent). This study demonstrates heparin-induced platelet activation in vivo in conditions of platelet-mediated thrombus formation. The observations are consistent with the concept of heparin-induced platelet activation as one of the mechanisms explaining the limited effects of heparin in preventing arterial thrombosis.

The effect of combined treatment with dextran 40 and acetylsalicylic acid on patency in severely traumatized small veins and arteries: an experimental study in the rabbit.

The effects of pharmacologic intervention on the fates of severely traumatized small veins and arteries have been studied in a rabbit model. Controls were given bolus doses of saline and a group treated with a combination of dextran 40 and acetylsalicylic acid starting prior to traumatization and continuing until postoperative day 5. Relative to controls, bleeding times in the treated group were significantly lengthened in arteries but not in veins, and venous patency significantly improved throughout the interval ending 2 weeks postoperatively. Arterial patency was at first highly improved but by 2 weeks, occlusion was virtually 100 percent. Since some of the occlusions took place more than a week after traumatization, the effects of antithrombotic agents on patency may need to be evaluated over considerably longer time periods than has previously been the rule.

The effect of dextran 40 on patency following severe trauma in small arteries and veins.

Arteriotomy/intimectomy and venotomy/intimectomy were performed in the rabbit ear. Low molecular-weight dextran (dextran 40) was infused 2 h before reperfusion and on postoperative days 1, 3 and 5 using a standard clinical protocol. Bleeding-times at reperfusion were recorded and patencies determined at intervals up to 2 weeks. Rabbits given single preoperative bolus doses of saline were used as controls. Dextran significantly prolonged bleeding-times in arteries and significantly improved early patency in both types of vessel, but the enhancements disappear by one week. Dextran 40 infusion thus has little effect on long-term patency.

Effects of the prostacyclin analogue iloprost on patency in small arteries and veins: an experimental study in the rabbit.

Arteriotomy/intimectomy and venotomy/intimectomy were performed in the ears of 43 rabbits. Twenty were treated with iloprost given as intravenous doses of 10 micrograms/kg body weight (bw) administered shortly before reperfusion followed by hourly infusions (3 micrograms/kg b w) until 12 hrs after reperfusion. At reperfusion venous and arterial bleeding times were noted. Patency was determined at 15-min intervals until 2 hrs after reperfusion and at 1 and 2 weeks postoperatively. As controls, 23 rabbits were given a single infusion of saline. Compared to controls, iloprost significantly prolonged arterial and venous bleeding times and significantly improved patency 2 hrs after reperfusion. One and two weeks later, however, virtually all vessels were occluded. Administered in this fashion, iloprost does not improve long-term patency in highly traumatized small veins and arteries.

Platelet accumulation and thrombus formation after microarterial injury. An experimental study in rabbits.

Patency rats and accumulation of 32P-labelled platelets were studied in the central ear arteries of rabbits (which were not treated with antithrombotic agents) after three types of vascular injury: End to end anastomosis, arteriotomy and superficial injury to the vessel wall to expose the lamina elastica interna/juxtaluminal parts of the tunica media, arteriotomy and deep injury to the vessel wall to expose the deeper layers of the tunica media. The superficial and deep vessel injuries were 5 mm long. Patency rates were 100% after end to end anastomosis and superficial injury, and 48% after deep injury. In a separate group of vessels with deep injuries the time course of formation of occlusive thrombi was investigated: occlusion was already present 15 minutes after reperfusion in all but one of seven occluded vessels. Platelet accumulation ratios were significantly higher after deep injury than after end to end anastomosis or superficial injury. In deeply injured patent vessels, platelet accumulation reached a maximum after about 30 minutes, which was later followed by a gradual decrease. Platelet accumulation patterns indicating sustained thrombogenicity throughout the measurement interval (embolization/reaccumulation patterns or late increases in accumulation) were encountered in only three of 22 deeply injured vessels. We conclude that: to cause formation of occlusive thrombus in otherwise healthy arteries and animals, a deep injury to the tunica media is necessary, and following reperfusion after repair of damaged vessels the time course of the thrombotic challenge is short.

Effects of streptokinase and urokinase on microarterial thrombosis and haemostasis. An experimental study in rabbits.

The effects of streptokinase and urokinase on haemostasis, accumulation of platelets radiolabelled with phosphorus (32P) and patency were studied after arteriotomy and deep vessel wall trauma of the central arteries of rabbits' ears. In one study, 12 rabbits were given 1700 IU/kg body weight of streptokinase or urokinase as intraaortic bolus injections five minutes before vascular reperfusion (opening of vascular clamps). A further six were given saline (controls). In the second, 12 further rabbits were each given 3,400 IU/kg of either substance, one fifth as a bolus before reperfusion and the remainder as a continuous infusion during the next two hours. A further six were given saline (controls). Irrespective of the dosage regimens, neither substance improved patency compared with saline-treated controls. Separate dose response studies with streptokinase showed that bolus or bolus+infusion doses larger than those given caused troublesome arteriotomy bleeding. Compared with controls, streptokinase increased, and urokinase decreased, accumulation of platelets. This was not reflected in differences in patency rates, which were similar in all groups. In conclusion, fibrinolytic stimulation with non-thrombolytic doses of streptokinase or urokinase did not prevent microarterial thrombosis in rabbits. The therapeutic index for these substances in clinical microvascular surgery is probably low, as haemorrhagic complications may be expected.

A comparison of the early antithrombotic effects between low molecular weight heparin and heparin in small arteries following a severe trauma: an experimental study.

Twenty-seven rabbits divided into 4 groups (2 control groups) received saline, standard heparin (400 IU both as anti-F Xa and activated partial thromboplastin time per kg of body weight), or low molecular weight heparin (lmwh) (Fragmin 560 IU as anti-F Xa and 140 IU as APTT units per kg of body weight). The figures represent the total dose given over 3 hours. The central arteries of the rabbits' ears were prepared and positioned in clamps. Platelets labeled with 32P were injected. Arteriotomy/intimectomy was performed. After reestablishment of blood flow, arteriotomy bleeding times, platelet accumulation, patency, and weight of thrombotic materials were measured. The bleeding time in the Heparin Group was significantly prolonged compared with its control group. But the bleeding times between the LMWH Group and the control group were not significantly different. The patency rates were increased in both the Heparin Group (100%, p < 0.01) and the LMWH Group (73%, p = 0.078) compared with respective control groups (43-50%). The mean weights of thrombotic material in each artery were significantly lower in the Heparin Group and in the LMWH Group than in their control groups. The mean values of radioactivity in all groups increased up to 15 minutes after the vessels were reperfused. There were no statistical differences between the treated groups and control groups. It was concluded that standard heparin is a very powerful inhibitor of thrombosis during the most crucial hours after reperfusion of severely traumatized small arteries, without significant effects on primary platelet adhesion/aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of microvascular thrombosis with low-dose tissue plasminogen activator.

In a blind, randomized study, two groups, each of seven rabbits, were treated with either a very low dose of human melanoma cell line-derived tissue-type plasminogen activator (t-PA) or isotonic saline. t-PA (0.067 mg/kg of body weight) was administered intraaortically, 20 percent being given as a 30-second "bolus" infusion just prior to the reperfusion of intimectomized central ear arteries and the rest as a continuous infusion during the next 2 hours. Arteriotomic bleeding times, accumulations of 32P-labeled platelets, patency, and sizes of thrombus deposits 2 hours after reperfusion were recorded. To confirm the presence of tissue plasminogen activator in plasma, fibrin-plate lysis assays of arterial plasma were performed immediately before and 1/2 hour and 2 hours after starting drug infusion. Arteriotomic bleeding times were similar in both groups. Transient "oozing" from wound edges occurred in 40 percent of rabbits treated with tissue plasminogen activator. Patency was significantly increased and thrombus deposits were smaller in the tissue plasminogen activator group. Plasma from animals treated with tissue plasminogen activator caused massive lysis of fibrin plates, whereas plasma from control animals caused little or no lysis. Platelet accumulations were very similar in both groups, indicating that occlusive thrombi mainly consisted of other elements than platelets (e.g., fibrin and red cells). Scanning electron microscopy showed normally adhering and aggregating platelets in both groups. This study shows that mild fibrinolytic stimulation with tissue plasminogen activator significantly improves patency in severely traumatized small-caliber arteries and indicates that such treatment may be one approach to prevent thrombosis at microvascular anastomotic sites.

Acute thrombogenicity of collagen coating of dacron grafts: an experimental study in sheep.

The early stages of platelet accumulation in two types of sealed interposition Dacron grafts implanted in the carotid arteries of sheep have been studied. One type was externally coated with collagen (Haemaguard) while the other experimental conduit had an additional inner lining of the same substance. 32P-labelled platelets were used to assess platelet accumulation and corrections for wall absorption were calculated. The activities in both types of grafts were highest proximally and decreased towards the distal anastomosis. The increases in the doubly-sealed grafts were larger than in those that had been single sealed, presumably reflecting activation of platelets in contact with collagen at the graft-wall and bloodstream interface. In addition, a significantly larger amount of thrombus was formed in the doubly-sealed grafts 4 h after reperfusion. It is reasonable to assume that increased acute thrombogenicity due to direct collagen-blood contact on graft surfaces is unfavourable to long term patency.

Collagen and gelatin-impregnated vascular grafts--is their thrombogenicity enhanced? An experimental study.

The early thrombogenicity of dacron vascular grafts with and without impregnation with collagen (n = 7) or gelatin (n = 7) was evaluated in a sheep carotid artery interposition model. Autologous platelets were labelled with 32P and reinjected. Flow was reduced to 25 ml/min to mimic a situation with poor run-off and the graft thrombogenicity was studied for 4 hours. The results showed no difference as regards patency, thrombus-free surface, or uptake of 32P-labelled platelets between collagen- or gelatin-impregnated grafts and their parent dacron grafts. Thus, no difference in early graft thrombogenicity could be documented.

Adverse effects of topical prostacyclin application in microvascular surgery: an experimental study.

The efficacy of topical prostacyclin as an antithrombotic agent was tested in a model of microvascular trauma. Preliminary measurements were made to determine a suitable dosage. Twenty-seven central arteries of rabbit ears were then prepared and 32P-labelled platelets infused intraaortically. Arteriotomy (7 mm) was followed by intimectomy (5 mm). Fifteen vessels in a control group were irrigated with Ringer's lactate and 12 vessels in an experimental group were treated with prostacyclin (10 ng/ml) in normal saline. Bleeding times at the sites of arteriotomy intimectomy, in vivo accumulations of isotope-labelled platelets, amounts of red thrombotic material, and patency were recorded. Patency was lower following prostacyclin treatment (1/12 as against 5/15) but not significantly so, and there were no statistically significant differences in other parameters. Prostacyclin treatment decreased vessel wall tone, interfering with blood-flow and promoting thrombus formation.

Importance of fibrinolysis in limiting thrombus formation following severe microarterial trauma: an experimental study in the rabbit.

In a blind randomized study, two groups of six rabbits were treated with either the fibrinolytic inhibitor tranexamic acid, 14 mg/kg bw, or isotonic saline solution (control group) given intraaortically as single bolus injections 5 min prior to arteriotomy and intimectomy of central ear arteries. Arteriotomic bleeding times, accumulations of 32P-labeled platelets, patency, and sizes of thrombus deposits 2 hr after reperfusion were recorded. Fixed vessels were observed by scanning electron microscopy. Bleeding times were similar in the two groups. The patency rate in the tranexamic acid group was 2/12, i.e., a significant reduction (P less than 0.05) from 7/12 in the control group. Thrombus deposits in occluded vessels contained large amounts of fibrin and red cells. Platelet accumulations in occluded vessels were significantly lower in the tranexamic acid group than in the control group, which indicates that the ratio of fibrin to platelets was increased in thrombi formed during antifibrinolytic treatment. This study has demonstrated the importance of normal fibrinolytic capacity in limiting thrombus formation following microarterial trauma. It is suggested that the use of antifibrinolytic agents in microvascular surgery should be restricted.

Effect of low and ultra low oral doses of acetylsalicylic acid in microvascular surgery. An experimental study in the rabbit.

About 10 h after administering acetylsalicylic acid (ASA) orally in doses of 4 mg and 20 micrograms/kg b.w., the central arteries of rabbit ears were subjected to severe vascular trauma (arteriotomy/intimectomy). Bleeding times from the trauma regions at reperfusion were measured and the activities from accumulating 32P-labelled homologous platelets recorded until 2 h after reperfusion when patencies were determined. In other studies, the effects of ASA on ex vivo platelet aggregation (aggregometry), thromboxane production, euglobulin clot lysis time and bleeding time following arterial puncture were investigated. Relative to controls, the following parameters were changed: patency was increased, as were the bleeding times following arterial puncture and thromboxane production was reduced. The median values of platelet accumulation were lower, but the changes were not statistically significant. Aggregometry showed decreased rates of platelet aggregability following treatment with ASA 4 mg/kg.

Studies of the antithrombotic effects of dextran 40 following microarterial trauma.

The effects of dextran 40 on platelet function and thrombus formation have been studied in vivo in arteries of the rabbit ear. Intra-aortic infusions of 32P-labelled platelets were followed by infusions of 1.7 g dextran 40 in 17 ml saline/kg b w. Treated and untreated groups were studied using as trauma either end-to-end anastomosis or arteriotomy (7 mm)/intimectomy (5 mm). After restoring blood flow, bleeding times at the sites of anastomosis and arteriotomy/intimectomy were recorded. Accumulations of labelled platelets were followed in vivo for 2 hours, after which patencies were determined and amounts of red thrombotic material evaluated. In a separate series of measurements, haematocrit levels after dextran infusion were studied. Nearly all vessels in the end-to-end anastomosis groups were patent. In the arteriotomy/intimectomy groups, there was a significant increase in patency following dextran infusion. Dextran infusion did not alter platelet accumulation following end-to-end anastomosis, but following arteriotomy/intimectomy median values were somewhat reduced. Its antithrombotic effects must therefore arise mainly at stages of thrombus formation subsequent to platelet deposition.

Hydroxyethyl starch increases patency and reduces thrombus formation following arteriotomy/intimectomy in small arteries: an experimental study in the rabbit.

Twenty-four arteries of rabbit ears, divided into two groups of 12 vessels each, were prepared and 32P-labelled platelets were infused. Arteriotomy/intimectomy was performed after 1 hr and in vivo platelet accumulation recorded for 2 hr. Group A comprised untreated control animals and group B was treated with 1 g hydroxyethyl starch (HES), MW 450,000 in 17 ml saline/kg b.w. (Plasma-steril). Vessel bleeding-times were normal, patency was improved, and intraluminal thrombotic material was reduced after HES treatment. Initial in vivo platelet accumulation was rapid and reached similar levels in both groups. However, the platelet accumulation curves decreased more frequently following HES than in the control group. HES does not prevent platelet accumulation at trauma sites, but reduces the sizes of the thrombi formed and may enhance disaggregation/fibrinolysis.

Does dextran 40 reduce early graft thrombogenicity? An experimental investigation on patency and platelet deposition on prosthetic graft materials in sheep.

The mechanism by which dextran 40 reduces early graft thrombogenicity has not been fully elucidated. Dextran improves haemaodynamics, reduces platelet aggregation, alters fibrin formation and enhances thrombus lysis. In this experimental investigation on sheep using a low flow model, the thrombogenicity of various grafts was studied when either a dextran or saline infusion was given. Bilateral carotid interposition grafts with expanded polytetrafluorethylene (ePTFE) on one side and dacron on the other (random allocation) were inserted in 12 sheep. The sheep were randomly divided into two groups, one given a dextran infusion and the other saline. A tendency for improved graft patency was seen in the dextran 40 treated animals (P less than 0.05 at 3 h). However, platelet accumulation did not differ markedly between the dextran 40 and saline treated groups. On the other hand there was a clear reduction of platelet accumulation on ePTFE grafts compared to dacron grafts (P less than 0.01). A large part of the radioactivity measured from the dacron graft was located within the graft wall. Further studies to clarify the mechanism of action of dextran are needed.

Washout of vessels with heparin does not improve patency following severe microarterial trauma: an experimental study.

The antithrombotic effect of heparin used as a wash-out solution in small arteries has been evaluated following severe vessel trauma (arteriotomy/intimectomy). Twenty-three central arteries of rabbit ears were prepared and platelets labeled with phosphorus-32 injected intraaortically. The arteries were positioned in double vascular clamps and opened by arteriotomy, and intimectomy was performed. After reestablishing blood flow, arteriotomic bleeding times, platelet accumulation in vivo, patency, and the amounts of intraluminal red thrombotic material were recorded. The rabbits were divided into two groups. In group A (12 vessels) the vessel interior was flushed with 2 ml of saline or Ringer's lactate solution. Group B (11 vessels) was similar except that the vessels were flushed with 2 ml of heparin, 100 IU/ml, in Ringer's lactate solution. In the control group (A) the bleeding time was 5 +/- 2 min, while in group B bleeding was so profuse that extra sutures had to be used in 6 of the vessels. Although significantly fewer platelets accumulated in heparin-treated animals, the patency frequencies and amounts of thrombotic material observed in both groups were similar. The final volume of thrombus formed is thus not always proportional to the degree of platelet accumulation. Washout of small traumatized arteries with heparin is thus not beneficial.