Andreas Hillarp

Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays.

Summary.  Introduction: Rivaroxaban is an oral direct factor Xa inhibitor developed for prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary but the dose‐dependent effects on common reagents and assay procedures are largely unknown. Objectives: To investigate the effect of rivaroxaban on commonly used coagulation assays. Materials and Methods: Rivaroxaban was added to plasma from healthy subjects in the concentration range 0–1000 μg L−1 and analyzed using different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, fibrinogen and activated protein C (APC) resistance assays. Results: At an expected peak concentration of rivaroxaban in clinical use, the APTTs were almost invariably prolonged but at lower concentrations the effect was weak. The concentration needed to double the APTT varied between 389 ± 106 and 617 ± 149 μg L−1 for different reagents. The PT assays showed a marked degree of difference. In general, the Quick PT type assays were more sensitive compared with the Owren type PT assays. The results from antithrombin assays were dependent on the type of reagent, with the Xa‐based assay being sensitive for rivaroxaban with an estimated increase of 0.09 IU mL−1 per 100 μg L−1 rivaroxaban. There were only minor effects on fibrinogen assays based on thrombin reagents. The APTT‐based assay for APC resistance is affected in a dose‐dependent manner whereas an assay based on the activation of coagulation at the prothrombinase level was unaffected. Conclusions: Different assays, and even different reagents within an assay group, display variable effects by therapeutic concentrations of rivaroxaban.

Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays

Dabigatran is an oral, reversible thrombin inhibitor that has shown promising results in large clinical trials. Laboratory monitoring is not needed but the effects on common coagulation assays are incompletely known. Dabigatran was added to plasma from healthy subjects in the concentration range 0-1,000 μg/l and analysed using several reagents for activated thromboplastin time (APTT), prothrombin time (PT), fibrinogen, antithrombin, and activated protein C resistance. Typical trough concentrations are about 50 μg/l, peak concentrations 100-300 μg/l. At 100 μg/l all APTT-results were prolonged. The concentration required to double APTT ranged between 227 and 286 μg/l, the responses for all five reagents were similar. PT-reagents were much less affected with almost no samples above INR 1.2 at 100 μg/l. The effect was sample dilution dependent with PT Quick type more sensitive than PT Owren type methods. If a patient on dabigatran has prolonged APTT, >90 seconds, and Quick PT INR>2 or Owren PT INR>1.5 over-dosing or accumulation of dabigatran should be considered. Two of four fibrinogen reagents underestimated the fibrinogen concentration considerably at expected peak concentration. Methods based on inhibition of thrombin over-estimated the antithrombin concentration, but not Xa-based. The APC-resistance methods over-estimated the APC-ratio, which may lead to miss-classification of factor V Leiden patients as being normal. Different coagulation assays, and even different reagents within an assay group, display variable effects at therapeutic concentrations of dabigatran. Some of these assay variations are of clinical importance, thus knowledge is needed for a correct interpretation of results.

Protein S binding in relation to the subunit composition of human C4b-binding protein

The human regulatory complement component C4b‐binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K‐dependent protein S. The major form of C4BP is composed of 7 identical, disulfide‐linked 70 kDa subunits (α‐chains), the arrangement of which gives the C4BP molecule a spider‐like appearance. Recently, we identified a unique 45 kDa subunit (β‐chain) in C4BP. We have now isolated a subpopulation of C4BP, which does notbind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the β‐chain. Another lower molecular weight form of C4BP was also purified. It contained the β‐chain and was efficient in binding protein S. Its subunit composition was judged to comprise six α‐chains and one β‐chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis‐a‐vis subunit composition and/or protein S binding ability and provide support for the concept that the β‐chain of C4BP contains the single protein S binding site.

Factor V leiden and prothrombin gene mutation: risk factors for osteonecrosis of the femoral head in adults.

The purpose of the current study was to determine whether factor V Leiden and the prothrombin 20210A gene mutation are risk factors for osteonecrosis of the femoral head in different etiologic groups of osteonecrosis in adults and whether patients with idiopathic osteonecrosis of the femoral head have a higher frequency of thromboembolic events compared with the general population. We investigated 63 adult patients with nontraumatic osteonecrosis of the femoral head for etiologic factors, such as corticosteroid medication and alcohol abuse, and the occurrence of factor V Leiden and the prothrombin 20210A gene mutation. In 35 patients, the disease was considered idiopathic and 10 of these patients (29%) had factor V Leiden or the prothrombin 20210A gene mutation or both. Mutations in factor V or the prothrombin 20210A gene were significantly more frequent in patients with idiopathic osteonecrosis than in a population of healthy control subjects (odds ratio, 2.7; 95% confidence interval range, 1.2–5.8) and in patients with osteonecrosis caused by corticosteroid medication or alcohol abuse (odds ratio, 10.8; 95% confidence interval range, 1.4–84). 36% of patients with a gene mutation had had a thromboembolic event compared with 8% of patients without a gene mutation. Thromboembolic events were more common among patients with idiopathic osteonecrosis (17%) compared with the general population (4%) and with patients with osteonecrosis caused by corticosteroid medication or alcohol abuse (7%).

GUIDELINES ON PREPARATION, CERTIFICATION, AND USE OF CERTIFIED PLASMAS FOR ISI CALIBRATION AND INR DETERMINATION

See also van den Besselaar AMHP, Barrowcliffe TW, Houbouyan-Re veillard LL, Jespersen J, Johnston M, Poller L, Tripodi A on behalf of the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the ISTH. Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination. J Thromb Haemost 2005; 2: 1946–53; van den Besselaar AMHP. Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination – reply to a rebuttal. This issue, pp 2371–2.

Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays

Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information.

A comparative in vitro evaluation of six von Willebrand factor concentrates

Summary.  The efficacy of von Willebrand factor (VWF) concentrates for treatment of von Willebrand disease (VWD) is dependent on their content of VWF and factor VIII (FVIII).

Resistance to activated protein C, the FV:Q506 allele, and venous thrombosis

Abstract Vitamin K-dependent protein C is an important regulator of blood coagulation. After its activation on the endothelial cell surface by thrombin bound to thrombomodulin, it cleaves and inactivates procoagulant cofactors Va and VIIIa, protein S and intact factor V working as cofactors. Until recently, genetic defects of protein C or protein S were, together with antithrombin III deficiency, the established major causes of familial venous thromboembolism, but they were found in fewer than 5–10% of patients with thrombosis. In 1993, inherited resistance to activated protein C (APC) was described as a major risk factor for venous thrombosis. It is found in up to 60% of patients with venous thrombosis. In more than 90% of cases, the molecular background for the APC resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine (R) at position 506 by a glutamine (Q). Mutated factor V (FV : Q506) is activated by thrombin or factor Xa in normal way, but impaired inactivation of mutated factor Va by APC results in life-long hypercoagulability. The prevalence of the FV : Q506 allele in the general population of Western countries varies between 2 and 15%, whereas it is not found in several other populations with different ethnic backgrounds. Owing to the high prevalence of FV : Q506 in Western populations, it occasionally occurs in patients with deficiency of protein S, protein C, or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting severe thrombophilia to be a multigenetic disease.

Prospective Longitudinal Study of Thromboelastography and Standard Hemostatic Laboratory Tests in Healthy Women During Normal Pregnancy

BACKGROUND:Hemostatic disorders are common in obstetric complications. Thromboelastography (TEG®) simultaneously measures coagulation and fibrinolysis within 10 to 20 minutes. Our primary aim in this prospective longitudinal study was to obtain knowledge about physiological changes in TEG® variables during normal pregnancy and 8 weeks postpartum. The secondary aims were to compare TEG® variables during pregnancy with TEG® variables 8 weeks postpartum and gestational weeks 10 to 15 and to correlate TEG® variables to standard laboratory analyses. METHODS:Blood samples were collected from 45 healthy pregnant women at gestational weeks 10 to 15, 20 to 22, 28 to 30, and 38 to 40, and at 8 weeks postpartum. The following TEG® analyses were performed: time until start of clotting (TEG®-R), time until 20-mm clot firmness (TEG®-K), angle of clotting (TEG®-Angle), maximum amplitude (TEG®-MA), and lysis after 30 minutes (TEG®-LY30). Activated partial thromboplastin time, prothrombin time, soluble fibrin, antithrombin, D-dimer, and platelet count were analyzed. RESULTS:Compared to 8 weeks postpartum TEG®-R was at least 0.9 minutes shorter (upper limit 99% confidence intervals) until gestational weeks 28 to 30 and the mean reduction varied between 23%–26%. TEG®-K was at least 0.1 minutes shorter throughout pregnancy and the mean reduction varied between 18%–35%. TEG®-Angle was at least 2.5 degrees greater during pregnancy and the mean increase varied between 12%–20%. TEG®-MA was also at least 0.4 mm greater during pregnancy and the mean increase varied between 6%–8%. TEG®-LY30 was at least 0.03% lower during gestational weeks 28 to 30 and 38 to 40 and the mean reduction varied between 67%–73%. The routine coagulation laboratory values were within normal pregnant limits. There were no or weak correlations between TEG® and the laboratory variables. CONCLUSIONS:TEG® demonstrates increased coagulability and decreased fibrinolysis during pregnancy. There was a faster initiation of hemostasis, with a minor increase in clot strength. Fibrinolysis decreased during late pregnancy. Alternative cutoff limits for TEG® variables may be required during pregnancy. Standard hemostatic laboratory tests were as expected during pregnancy. Future studies are needed to ascertain whether viscoelastic methods are preferable to standard hemostatic tests for the diagnosis of coagulopathy during obstetric hemorrhage.

Factor V Leiden and the prothrombin 20210A gene mutation and osteonecrosis of the knee.

IntroductionThe pathogenesis behind osteonecrosis of the knee is still unknown. Circulatory impairment of the bone secondary to thrombosis in the microcirculation has been suggested as a mechanism. The purpose of this study was to examine the association between osteonecrosis of the knee and abnormalities in the thrombotic pathway in the form of factor V Leiden and the prothrombin 20210A gene mutation.Materials and methodsThirty-eight consecutive patients (13 men and 25 women) with osteonecrosis of the knee without a history of knee trauma or surgery to the knee were enrolled in this study. Assays for the detection of factor V Leiden and the prothrombin 20210A gene mutation were performed, and the results were compared with those from 282 healthy volunteers.ResultsSix patients were diagnosed with secondary osteonecrosis, four corticosteroid-induced and two alcohol-induced. In 32 patients, no aetiological factor was found, and these patients were diagnosed with primary osteonecrosis of the knee. Twelve patients had 14 gene mutations, 11 factor V Leiden and 3 prothrombin 20210A gene mutations. Factor V Leiden and the prothrombin 20210A gene mutation occurred significantly (p=0.006) more frequently in patients with osteonecrosis than in a population of 282 healthy volunteers (odds ratio 3.1, 95%CI 1.4–6.6).ConclusionThe results of this study suggest that coagulation abnormalities in the form of factor V Leiden and the prothrombin 20210A gene mutation might play a role in osteonecrosis of the knee.