Heparin was purified from gills and intestines from farmed Atlantic salmon (Salmo salar). Heparin activity was determined after size exclusion chromatography in the molecular weight range from above 8,000 to near 1,500. A specific activity of 110.1 antifactor Xa units/mg was measured in the less than 3,500 molecular weight fraction while 136.8 antifactor Xa units/mg was detected in a 8,000-3,500 molecular weight fraction. The presence of high affinity salmon heparin was demonstrated by using chromatography on antithrombin-Sepharose. Heparin with molecular weights lower than 3,500 was found both in high and low affinity fractions. NMR-analysis detected N- and O-sulfated oligosaccharides essential for heparin activity. The amount of salmon heparin with molecular weight lower than 8,000 varied from 12% to almost 100%. The factors determining this variation is not known, but appears to reside in the fish at the time of slaughter. The in vivo effect of salmon heparin was tested in rabbits using dalteparin as control. Salmon heparin activity was recovered in plasma samples expressed as antifactor Xa activity after intravenous administration. Based on a small number of samples and animals, the results indicate that in vivo half-life time of salmon heparin was higher than that of dalteparin.

The therapeutic effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in postmenopausal osteoporosis was tested in a single blind, randomized prospective study. Thirty-nine women, 50-65 years of age, were treated for three years with 0.5 microgram 1,25(OH)2D3 daily. In a control group, 37 women were given 400 IU vitamin D3 daily. There was no significant difference in annual bone loss from the distal or proximal forearm between the groups. New vertebral fractures were evaluated, and in the treatment group, the annual increase in vertebral fractures was 0.18 +/- 0.387 and in the control group 0.13 +/- 0.330. New long bone fractures were 7 and 5, respectively. None of the observed differences were statistically significant. In the 1,25(OH)2D3 group, 28% had to reduce the dose because of slight hypercalcaemia. We conclude that 1,25(OH)2D3 as used in this study is not effective in the treatment of osteoporosis.

We have evaluated an automated, simplified, turbidimetric method for the measurement of the C4b binding protein (C4bBP). A comparison with a manually performed electroimmunoassay in plasma samples referred for coagulation analysis (n=80) revealed a correlation coefficient of 0.88. Lipaemic plasma is not suitable for analysis, whereas moderately haemolytic or icteric plasma may be used with the present method. In young and middle-aged patients (n=33) investigated 3 or more months after an episode with thrombosis of unknown reason, the mean C4bBP concentration was not significantly different from the mean found in healthy controls (n=38). This result is in accordance with the hypothesis that C4bBP is an acute phase reactant. The results indicate that the turbidimetric assay may replace the electroimmunoassay in clinical work.

BACKGROUND:Free testing, treatment and extensive information campaigns are used to monitor and control the incidence of Chlamydia trachomatis infections in Norway. Most programmes have 15 to 25 year-olds as their target, because of the high incidence of infection in this age group. The potential role and effect of internet-based commercial testing has not previously been assessed in this context.MATERIAL AND METHODS:1458 urine samples, taken by the patients themselves, were collected from March 2005 to September 2006 according to instructions given on the commercial web site www.testselv.no, and sent to a given address for analysis. Sex, age distribution and prevalence of Chlamydia trachomatis infection were assessed and all costs were paid by the patient buying the service.RESULTS AND INTERPRETATION:More men than women used this service, in contrast to the sex distribution seen in public screening programs. The mean age was 28 years, the 25 % percentile and the 75 % percentile was 24 and 32 years, respectively. The prevalence of infection was high; 7.5 % in women and 12.5 % in men. Our study identifies a demographic group with a high incidence of Chlamydia trachomatis infection that has not been previously been targeted by public screening programmes.

Three global assays, the Calibrated Automated Thrombogram (CAT), the ProC Global (PCG), and the Coagulation Inhibitor Potential (CIP) were performed in frozen plasma samples from 24 normal controls and 24 patients with inherited thrombophilia. Six patients had inherited antithrombin (AT) deficiency; 18 patients had abnormalities in the protein C/S anticoagulant system (protein C deficiency (n=3), protein S deficiency (n=10), homozygous FV Leiden mutation (n=5)). Nine of these twenty four patients carried additionally the heterozygous FV Leiden mutation. All three assays separated the thrombophilia group and the control group (P=0.083 for CAT, P<0.0001 for the other two assays) but there was considerable overlap, particularly in the CAT assay. The CAT assay separated all plasma samples with AT deficiency but was less sensitive to abnormalities in the protein C/S system. In contrast, ProC Global was more sensitive to abnormalities in the protein C system than to AT deficiency. The CIP assay was approximately equally sensitive to defects in both systems. Receiver operator characteristic (ROC) curves confirmed that the ProC Global and the CIP assays performed better than the CAT assay (P=0.0179 and P=0.0003, respectively). With the CIP assay ROC analysis showed that with a sensitivity of 100% the specificity was 87.5%. With the PCG assay, optimal threshold resulted in both a sensitivity and a specificity of 79.2%. Although our material is relatively small, the data suggest that at a cut-off value with a specificity of >80%, the CIP assay should be evaluated as a screening test for severe thrombophilia.

Summary Prothrombin time (PT) is clinically important and is used to monitor oral anticoagulant therapy. To obtain PT results in international normalized ratio (INR), the current standardization procedure is complex and involves reference reagents.The PT of diluted plasma samples can be determined with a combined thromboplastin (the Owren-type procedure), but not necessarily with a plain thromboplastin (the Quick-type procedure). Owren-type PT procedures can therefore, as an alternative to the INR calibration, be calibrated with diluted normal plasma to give PT results in percent of normal PT activity (PT%).The present study explored if a plasma-based calibration of an Owren-type PT procedure can be used to obtain results in INR. The approach was to establish a relationship between PT% and INR by multi-center analysis of 365 samples from healthy individuals and patients on warfarin treatment. INR values were obtained by manual Quick-type reference procedure and PT% values by various automated Owren-type procedures. A relationship INR = (1/PT% + 0.018)/0.028 was found. A calibration procedure, based on the relationship, was investigated. Calibrators were the median PT of 21 normal plasma at dilutions representing 100%, 50%, 25%, 12.5% and 6.25% of normal PT activity. These were assigned INR values of 1.00, 1.36, 2.07, 3.05 and 6.36. Calibration of various Owren-type assays was repeatedly performed by 5 expert laboratories during 3 consecutive years. The INR values of certain lyophilised or frozen control plasmas were determined. The frozen control plasmas had externally assigned INR values according to WHO guidelines. Within the laboratory, CV was typically below 3%. No appreciable difference among the results of the different laboratories or the three assay occasions was found. Externally assigned and INR values were essentially identical to those found. These and other results indicated that the calibration procedure was reproducible, precise and accurate. Thus, an Owren-type PT assay can be calibrated with normal plasma samples to give results in INR and the investigated calibration procedure can be proposed for this purpose.

ABSTRACTThrombin from Atlantic salmon (Salmo salar) was purified and characterized as a potential new binding agent for the food industry. Purification was performed avoiding inhibitors, using BaSO4 adsorption and heparin‐Sepharose affinity chromatography. Prothrombin activation was performed using a mixture of eggs and gills from salmon. Optimized conditions for the adsorption, elution, and the activation step are presented. The purified thrombin clotted bovine fibrinogen with a specific activity of 1423 U/mg. Sequence data are presented and compared with other species. This method of nontoxic activation and purification will allow salmon thrombin to be used in the food industry.

Lipoprotein and hemostatic profiles including coagulation inhibitors were determined in 136 patients with acute ischemic stroke. Based on clinical examination, cerebral computed tomography, Doppler ultrasonography of precerebral arteries and transthoracic echocardiography, the strokes were classified as cardioembolic (n = 38), non-cardioembolic (n = 92), and mixed cardioembolic/hypertensive (n = 6). Patients with cardioembolic stroke were older than patients with non-cardioembolic stroke. Lipoprotein(a) was higher in the cardioembolic than in the non-cardioembolic group. Lipoprotein(a) was not significantly correlated to the other lipid levels and may represent an independent lipid risk factor. The non-cardioembolic group had higher levels of total cholesterol, triglycerides, total cholesterol/high-density lipoprotein cholesterol ratio, low-density lipoprotein cholesterol, apolipoprotein A1, and apolipoprotein B. The cardioembolic group had higher concentrations of fibrinogen and D-dimer, and lower levels of antithrombin, protein C, protein S and heparin cofactor 2 than the non-cardioembolic group. The differences in the hemostatic profile are consistent with thrombosis due to activated coagulation being more involved in the pathogenesis of cardioembolic than of non-cardioembolic stroke. Lipoprotein(a) seems to be more associated with coagulation markers of thrombosis than with atherosclerosis, whereas the other lipids mainly seem to be risk factors for atherosclerosis.

Evidence is presented, confirming the presence of an anticoagulant system in the plasma of Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss Walbaum) (Order: Teleostei, Family: Salmonidae) that bears striking similarities with the protein C anticoagulant system in mammals; its vitamin K-dependence was documented through a warfarin feeding trial. A potent activator of this system is the protein C activator from the venom of the Central American Moccasin, Agkistrodon bilineatus. When activated, the system splits the tripeptide substrate glu-pro-arg-pNa, which is the substrate preferred for the in vitro assay of human protein C. It also prolongates the plasma activated partial thromboplastin time, indicating that the system is of clinical relevance. A temperature dependence of the plasma protein C-like activity was evident, the mean activity being 5- to 10-fold higher, but also more variable, in both species in summer and early fall, than it was in winter. There was also a species difference, with rainbow trout having the higher levels. In man, subnormal values of protein C implies an increased risk of thrombosis. Whether this applies to fish is not known. It is, however, a fact that microvascular thromboses are prevalent in farmed Atlantic salmon in winter, whereas thrombotic disease is not a problem in rainbow trout; in the present study plasma protein C-like activity was 30% (of a human reference plasma) in salmon at 4 degrees C compared to a level of 60% in rainbow trout. A complicating factor for the assay of protein C-like activity in salmonid. plasma, is the poor stability of the inhibitory system upon storage. Consequently, assays have to be done with freshly prepared citrated plasma.

Journal of Fish DiseasesVolume 18, Issue 6 p. 623-626 Evidence of ongoing coagulation and fibrinolysis in early-maturing Atlantic salmon, Salmo salar L. R. SALTE, Corresponding Author R. SALTE AKVAFORSK (Institute of Aquaculture Research Ltd., Ås.Dr Ragnar Salte, AKVAFORSK (Institute of Aquaculture Research Ltd), P.O Box 5010, N-1432 Ås. Norway.Search for more papers by this authorK. NORBERG, K. NORBERG AKVAFORSK (Institute of Aquaculture Research Ltd., Ås.Search for more papers by this authorO. R. óDEGAARD, O. R. óDEGAARD Department of Clinical Chemistry, Aker Hospital, Oslo, NorwaySearch for more papers by this author R. SALTE, Corresponding Author R. SALTE AKVAFORSK (Institute of Aquaculture Research Ltd., Ås.Dr Ragnar Salte, AKVAFORSK (Institute of Aquaculture Research Ltd), P.O Box 5010, N-1432 Ås. Norway.Search for more papers by this authorK. NORBERG, K. NORBERG AKVAFORSK (Institute of Aquaculture Research Ltd., Ås.Search for more papers by this authorO. R. óDEGAARD, O. R. óDEGAARD Department of Clinical Chemistry, Aker Hospital, Oslo, NorwaySearch for more papers by this author First published: November 1995 https://doi.org/10.1111/j.1365-2761.1995.tb00367.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume18, Issue6November 1995Pages 623-626 RelatedInformation

Antithrombin from bony fish (Teleostei), represented by an ancient salmonid, Atlantic salmon (Salmo salar L.), and a more evolved species from the same family, rainbow trout (Oncorhychus mykiss Walbaum), functions in vitro as does its human counterpart: it inactivates thrombin almost instantaneously in the presence of heparin and only slowly when heparin is absent. The inhibitory activity of salmonid antithrombin towards the homologous thrombin did not differ noticeably from its inactivating capacity in heterologous (teleost) systems, and enzyme-inhibitor reactions between reagents from fish and man proceeded just as efficiently. In all enzyme-inhibitor reactions with salmonid thrombin the inactivation by salmonid antithrombin or diluted fish plasma was maximal at pH 7.8 - 8.4. The inactivation was clearly dependent on heparin in all systems and maximal at concentrations between 1.5 and 6 U/ml. What particularly distinguishes the salmonid thrombin-antithrombin interaction from the human one is that the former has to function over a wide range of temperatures. And the thrombin inactivating capacity of purified antithrombin and diluted plasma in the presence of heparin was indeed present at temperatures down to 3 degrees C, a capacity that human antithrombin also has retained. Even more interesting was that the teleost enzyme-inhibitor reaction was nearly independent of temperature under the conditions studied.

Multifocal intestinal infarctions, due to thrombosis in small vessels, might be a pathogenetic mechanism for Crohn's disease (CD). Deficiency of free protein S may contribute to the development of such thrombotic occlusions. In the present study free protein S was measured in 54 patients with CD. In 31 patients (57.4%) the plasma concentrations of free protein S were below the lower normal range. The mean value of free protein S in CD patients was 72.2%, as compared with 97.5% in healthy subjects (p < 0.01). The concentrations of C4b-binding protein and protein C were similar in the two groups. Free protein S levels were not correlated to disease activity, previous surgery or complications, extraintestinal manifestations, or current medical therapy. The impairment of the protein S/protein C/thrombomodulin system found in patients with CD favours coagulation and might be of importance for both the development of CD and its thromboembolic complications.

Protein S deficiency is an autosomal dominant inherited disease. The authors describe a family in which 27 of the 52 members had a deficiency of S protein. Eight family members have suffered from thromboembolic disease.

Protein S deficiency increases risk of thrombosis. At present, we have information on 63 Norwegian individuals with hereditary protein S deficiency belonging to 25 different families. 42 of the individuals have experienced at least one thromboembolic episode, and seven a cerebral infarction before the age of 70 years. The amount of free protein S in plasma is dependent on variation of the acute phase protein C4b-binding protein (C4bBP). Acute phase response with increased C4bBP induces free protein S deficiency, and increases risk of thrombosis. In patients with protein S deficiency, warfarin may reduce free protein S to critically low levels, and thus explain why, in some patients, recurrent thrombosis occurs during warfarin treatment. In this situation, warfarin should be replaced by heparin.

Consumptive coagulopathy was induced within 4 h in Atlantic salmon, Salmo salar L., by injecting purified serine protease from Aeromonas salmonicida into the dorsal aorta. Pretreatment with a bolus intravascular injection of (human) antithrombin (AT) or (bovine) alpha2-macroglobulin (alpha2M) just prior to injection of the protease alleviated the in vivo pro-coagulant effects of the enzyme, but could not hinder the development of consumptive coagulopathy. In fish receiving only saline as pretreatment, the coagulopathy was evident even after 28 h, but the fish were not overtly sick. The addition of the exogenous inhibitors increased the fish's natural protection against the bacterial exotoxin, suggesting that both AT and alpha2M are of importance for the outcome of the pathologic process. Results further indicate that while AT in vivo was mainly directed against generated thrombin and activated coagulation factor X (FX(a)), alpha2M inhibited the protease directly.