Nils Egberg

Effects on the hemostatic system and liver function in relation to Implanon and Norplant. A prospective randomized clinical trial.

In this prospective randomized clinical trial, two long-term contraceptive implants were studied with respect to hemostasis and liver function in 86 healthy young women. The two implants used were Implanon, containing the progestagen etonogestrel (the biologically active metabolite of desogestrel) and Norplant, the implant containing the progestagen levonorgestrel. The results of the trial showed that both implants had similar small effects on the hemostatic system that are not suggestive of a tendency towards thrombosis. The effect on liver function was characterized by increases in total bilirubin and gamma-glutamyl transferase and decreases in alanine aminotransferase and aspartate aminotransferase.

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

Determination of vitamin K sensitive coagulation factors in plasma. Studies on three methods using synthetic chromogenic substrates

Abstract Methods used for monitoring coumarol treatment should be sensitive to the loss of calcium binding γ-carboxyl glutamic acids in the prothrombin or factor X molecules. In this study determinations of generated enzymatic activity has been performed by means of chromogenic oligopeptide synthetic substrates: Bz-Ile-Glu-Gly-Arg-pNA sensitive to factor X a and H-D-Phe-Pip-Arg-pNA sensitive to thrombin. Two activating principles have been investigated, Russel viper venom (RVV), and the venom from Echis carinatus (Ecarin). When a substrate specific for thrombin was used to measure the activity generated after RVV-lipid activation a method sensitive to factors II, V and X was obtained. When using RVV without lipid and a substrate specific for factor X a , however, the method was specific for factor X. These two methods were both sensitive to the coumarol induced defect and correlated well with the Thrombotest method when tested on coumarol plasmas (r = 0.85 and r=0.84, respectively). The activity generated by Ecarin was registered on a thrombin sensitive substrate. This method was specific to prothrombin but was less sensitive to the coumarol induced prothrombin defect and resulted in a noncorrelated relation to Thrombotest using coumarol plasma. It is suggested to use the factor X specific method for monitoring coumarol treatment. The specificity and reproducability of this method is better than for the clotting techniques. In addition this method is a more accurate enzymatic determination which is easily automized.

INR calibration of Owren-type prothrombin time based on the relationship between PT% and INR utilizing normal plasma samples

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 guide-lines. 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.

Original Research ArticlesEffects on the hemostatic system and liver function in relation to Implanon® and Norplant®1: A prospective randomized clinical trial

In this prospective randomized clinical trial, two long-term contraceptive implants were studied with respect to hemostasis and liver function in 86 healthy young women. The two implants used were Implanon, containing the progestagen etonogestrel (the biologically active metabolite of desogestrel) and Norplant, the implant containing the progestagen levonorgestrel. The results of the trial showed that both implants had similar small effects on the hemostatic system that are not suggestive of a tendency towards thrombosis. The effect on liver function was characterized by increases in total bilirubin and gamma-glutamyl transferase and decreases in alanine aminotransferase and aspartate aminotransferase.

Local INR calibration of the Owren type prothrombin assay greatly improves the intra- and interlaboratory variation A three-year follow-up from the Swedish national external quality assessment scheme

In 1999, a simplified procedure for calibration of the Owren prothrombin time (Owren PT) assay was introduced by a working group of the organisation for national quality assurance in laboratory medicine in Sweden. The new protocol allowed local calibration by means of only two lyophilised national plasma calibrators and expression of results as an international normalized ratio (INR). This is our report of a three-year follow-up involving the analysis of data from all laboratories, in hospitals (n=88 in 2002) and primary health care units (n=246 in 2002) that perform the Owren PT assay in Sweden. The interlaboratory variation was significantly improved after the introduction of the new calibration procedure. For the larger hospital-based laboratories, the mean coefficient of variation (CV) was reduced from 7.9% to 5.2% (p<0.0001) when analysing test materials with INR range 2-4. In the higher INR range (>4), the CV was reduced even further, from 10.4% to 6.8% (p<0.0001). The corresponding results from smaller laboratories in the primary health care units showed a similar decrease in CV from 8.2% to 5.7% in the INR range 2-4 (p<0.0001). At the INR range >4, the CV was reduced from 9.5% to 7.8%. The intralaboratory variation was also improved for both types of laboratory categories. This study shows an improved precision, with CV less than 6% at the therapeutic INR range, for both hospital-based laboratories and smaller laboratories in the primary health care system. The results indicate that the Owren PT assay is well suited for local INR calibration employing only two calibrant plasmas in a simplified procedure.

A global assay of haemostasis which uses recombinant tissue factor and tissue-type plasminogen activator to measure the rate of fibrin formation and fibrin degradation in plasma

The global assay of Overall Haemostasis Potential we previously described has been refined. The coagulation cascade in platelet-poor plasma is triggered by adding a minimal dose of recombinant tissue factor together with purified phospholipids and calcium; fibrinolysis is initiated by adding recombinant tissue type-plasminogen activator in a concentration similar to what can be obtained during thrombolysis. Numerical differentials of optical densities reflecting rates of fibrin formation and degradation are calculated by a new software, and the Coagulation Profile (Cp) and the Fibrinolysis Profile (Fp) are determined. The combined effect of these counteractive systems is expressed as a ratio of Cp to Fp, called the Overall Haemostasis Index. Commercially available coagulant-deficient patient plasma samples and plasma with various amounts of added PAI-1 are examined; changes of fibrin turbidity demonstrate that this assay can determine Cp and Fp in a physiologically relevant way. Increased Cp and decreased Fp in prothrombotic patients, as well as expected effects of heparin or a thrombin inhibitor on Cp and Fp, suggest that our method can detect hypercoagulability and assist in monitoring antithrombotic treatment. Ongoing studies will show whether this simple assay can be of value in clinical routine.

Experimentally induced stress in man: Effects on blood coagulation and fibrinolysis

Abstract Sixteen healthy human females were exposed to a 77-hr vigil under strictly controlled conditions. Blood coagulation factors V, VIII and IX and fibrinogen decreased significantly during the vigil. Only the latter had returned to preexposure values within 5 days after the vigil. No increased fibrinolytic activity was detected. Concomitant adrenal reactions, interpreted as indices of stress, are reported and discussed in relation to these results. It is concluded that dhe decrease of coagulation factor activity might be interpreted as an expression of adaptation to prolonged stressor exposure.

Transmission of HIV Infection to Heterosexual Partners but Not to Household Contacts of Seropositive Haemophiliacs

The prevalence of HIV antibodies in 44 heterosexual female partners and 56 nonsexual family household contacts of 61 HIV seropositive haemophiliacs (41 adults and 20 children or adolescents) was determined to evaluate the risk of transmission of HIV infection. HIV antibodies were determined by enzyme-linked immunosorbent assay and positive reactions were confirmed by Western blotting. HIV antibodies were demonstrated in 4/40 (10%) regular heterosexual partners of 40 seropositive patients with haemophilia A. Four temporary heterosexual partners of one additional seropositive haemophiliac were seronegative. 56 nonsexual household contacts including 30 parents, 13 siblings and 13 children of 29 seropositive haemophiliacs were all negative for HIV antibodies. Thus transmission of HIV occurred from seropositive haemophiliacs to their heterosexual partners but not to nonsexual household contacts.

Treatment with recombinant human erythropoietin induces a moderate rise in hematocrit and thrombin antithrombin in healthy subjects.

Recombinant human erythropoietin (EPO) therapy in uremic patients raises the hematocrit (Hct) and increases physical exercise capacity (1,2) and quality of life (1). In general, partial correction of anemia to subnormal levels in uremic patients has proven to be safe with few serious adverse effects apart from hypertension (3). Ever since the advent of EPO the prospect of abuse of the hormone by sportsmen has been subject to scrutiny. Both maximal oxygen uptake and endurance capacity are increased after EPO treatment in healthy subjects (4). Moreover, EPO treatment in healthy subjects has been found to induce an accentuated blood pressure reaction after submaximal exercise (5). Previous studies have shown that extreme physical exertion can predispose to an increased intravascular coagulation (6). Moreover there is a significantly increased risk of thrombosis in patients with myeloproliferative disorders, particularly in polycythemia vera (7). An enhanced risk of cardiovascular events may therefore arise should sportsmen abuse EPO as a blood doping agent. The aim of this study was to examine the effects of an EPO-induced increase in Hct on the coagulation system in healthy subjects.