Non-Compartment and compartmental pharmacokinetics, efficacy, and safety of Kedrion FIX concentrate.

BACKGROUND: An open-label phase II, multicenter clinical trial was conducted at 11  Haemophilia Centres in  Italy, Romania, and Turkey, to evaluate the pharmacokinetics (PK), efficacy, and safety of high purity, plasma-derived, double virus inactivated and double nano-filtered factor IX (pd-FIX) concentrate (Kedrion FIX), EudraCT Number: 2005-006186-14. MATERIAL AND METHODS: 16 previously treated patients (PTPs) with severe or moderately severe haemophilia B were enrolled in the study. At enrolment, 14 underwent the first PK assessment (PK I), and the second PK (PK II) assessment was performed after six months of treatment (5 on-demand and nine prophylaxis) at the end of the study. PK parameters were evaluated by Non-Compartmental Analysis (NCA), One-Compartment model (OCM), and Two-Compartment Model (TCM). Efficacy of Kedrion FIX in all 16 patients was evaluated by the number of bleeding events, and clinical response following the infusions. Periodic FIX inhibitor assays and thrombogenicity tests were scheduled throughout the study to assess the safety of the drug. RESULTS: As compared to the published data on PK of pdFIX, Kedrion FIX displayed a longer half-life (22.37-55.73 hrs), reduced clearance, and regular volume of distribution at PK I by both NCA and OCM. The comparison of outcomes of PK II with those of PK I by OCM,  also showed significant changes, particularly in patients on prophylaxis, who showed some improved parameters of PK. Due to two outlier values at the end of the trial, the NCA parameters of PK I were not compared to those of PK II. Breakthrough bleeds were successfully treated with 1 or 2 infusions. No significant adverse events were observed during the study. DISCUSSION: During the six-month clinical study period, the use of Kedrion FIX resulted in a safe and effective pd-FIX concentrate with excellent PK characteristics.

Tapering and discontinuation of thrombopoietin receptor agonists in immune thrombocytopenia: Real-world recommendations.

Thrombopoietin receptor agonists (TPO-RAs) are currently indicated for continuous treatment of chronic primary immune thrombocytopenia (ITP). However, there is growing evidence that TPO-RAs can also trigger sustained response in 10-30% of cases after treatment tapering and discontinuation. Therefore, at least for selected responding patients, it might be rational to plan TPO-RA interruption to exploit off-treatment response. Intriguingly, complete or partial responses with TPO-RAs are frequently observed when treatments are initiated early, suggesting that unknown immune-related mechanisms may be involved in this phenomenon. The sustained responses observed after interruption of TPO-RAs may be interpreted as a recovery of immunological tolerance; thus, the re-establishment of immunological equilibrium might be primarily responsible for the observed off-treatment effect. Importantly, these findings may indicate that anticipated TPO-RA usage can lead to improved responses, and that optimized tapering and interruption in selected patients can furthermore improve prognoses. On the base of this rationale, a series of real-life considerations have been generated by a panel of Experts to elucidate possible novel criteria and modalities to identify subgroups of patients who can benefit from tapering and/or discontinuation of TPO-RAs. Towards this aim, the results of a survey of ITP experts are herein reported, reflecting a snapshot of current real-life experience on early discontinuation of TPO-RA-based therapy. The present manuscript also highlights the importance of future translational studies on novel prognostic and predictive biomarkers that can stratify patients and facilitate the clinical choice for second-line treatment of ITP.

Characterization of Physicians Workload in a Reference Center for the Treatment of Thrombotic and Bleeding Disorders.

Heavy workloads in healthcare have been often associated to adverse clinical outcomes. To reduce workloads, an optimal scheduling of clinical staff resources is not enough, but particular attention must be payed to work organization and task characteristics. Moreover, interruptions during the clinical practice contribute to increase perceived workloads. In this study we analyzed and characterized the physicians' workload in an Italian center for the treatment of thrombotic and bleeding disorders. First, all clinical and administrative processes performed in the center were analyzed by means of two process modelling tools. Then, the quantification of the physicians' workload and the characterization of interruptions during practice were conducted. From our results it emerged that the task that mainly impacts on the workload is ambulatory care (42% of total workload) while interruptions produce a delay of almost 15 minutes per day and mainly occur during visits. Including all activities, the total daily workload per physician was 8 hours on average. In this time breaks were not taken into account. Concluding, from our analysis it is evident that the physicians' workload in the analyzed center is heavy and interruptions represent a source of delay in the workflow, that impact the physicians' workload.

Assessment of platelet function analyzer (PFA-100) in kidney transplant patients before renal allograft biopsy: a retrospective single-center analysis.

BACKGROUND: Kidney biopsy (KB) represents the criterion standard to obtain information on diagnosis and prognosis of renal allograft dysfunctions. However, it can be associated with bleeding complications (BCs). Bleeding time test (BTT), the best predictive indicator of post-biopsy BCs, is not a very reproducible test and is invasive. Therefore, the aim of this study was to evaluate whether the platelet function analyzer (PFA-100), a very reliable test to investigate primary hemostasis, could be useful in predicting the risk of bleeding complications in transplant patients undergoing KB. METHODS: We carried out a retrospective analysis of PFA-100 collagen-epinephrine (C-EPI) and collagen-adenosine diphosphate (C-ADP) closure times in 119 patients undergoing KB in our center. Data regarding BTT, age, sex, blood pressure, number of renal allograft punctures for each biopsy procedure, thromboplastin time, prothrombin time, complete blood count, and prophylactic therapy with desmopressin were also collected. Major (need for blood transfusion) or minor (no need for any intervention) BCs (hematoma and hematuria) were recorded. RESULTS: Indications for KB were: delayed graft function (n=23), allograft dysfunction (n=40), proteinuria (n=27), allograft dysfunction plus proteinuria (n=19), and protocol biopsy (n=10). Nine of the 119 patients (7.5%) developed minor BCs (6 macrohematuria, 3 hematoma), major BCs did not develop. No significant differences were found in any of the clinical and laboratory data, including BTT and PFA-100 (C-EPI and C-ADP) between patients who developed BCs compared with those who did not. In addition, there was no correlation between PFA-100 test (C-EPI and C-ADP) values and BTT data [R2=0.002; P=.6]. CONCLUSIONS: The PFA-100 test was not useful in predicting the risk of BCs in kidney transplant patients undergoing renal allograft biopsy.

Use of modified functional assays for activated protein C resistance in patients with basally prolonged aPTT.

Inherited resistance to activated protein C (APCr) is currently recognized as the most prevalent cause underlying venous thrombophilia, with an estimated prevalence around 20% in thrombotic patients and around 1.8-7% in the general population. A correct laboratory diagnosis of APCr is therefore essential. Two different diagnostic approaches are at present at our disposal: the semi-quantitative plasma test based on the measurement of two aPTTs (in the presence and absence of activated protein C), and the detection of the factor V Arg506 Gln mutation by DNA analysis. In this study we firstly evaluated sensitivity, specificity and diagnostic efficiency of an aPTT-based plasma clotting test (Chromogenix, Sweden) versus DNA analysis; then, since the APC resistance test is invalidated by a basally prolonged aPTT (i.e. during warfarin and heparin therapy or in patients with clotting factor deficiencies or in the presence of a lupus anticoagulant), patient plasmas were conveniently diluted in factor V deficient plasma in order to correct clotting factor abnormalities. Nevertheless, patients with a LA and an aPTT ratio range 1.8-3.17 were still all misclassified. We obtained correct diagnoses in LA positive patients by preincubating plasmas with a mixture of phospholipids; therefore we decided to perform a double modified clotting test adding a mixture of platelet derived phospholipids to samples previously diluted in factor V deficient plasma. The performance characteristics of this novel method with a different aPTT reagent (Behring, Germany) were also evaluated. With this double modified test all patients were correctly classified as negative or positive for factor V mutation in agreement with DNA analysis, irrespectfully of the basal aPTT value and the aPTT reagent employed. We propose this modified version of the APCr clotting test as an easily reproducible, reliable, very sensitive and specific screening test which possibly reduces the need for DNA analysis.

Natural and acquired inhibitors of hemostasis in selected symptomatic outpatients with venous thromboembolic disease.

BACKGROUND AND OBJECTIVE: Deficiencies of natural inhibitors and the presence of lupus anticoagulant are important risk factors leading to venous thromboembolic events. Before resistance to activated protein C (APC-R) was identified, the overall prevalence of inherited abnormalities of hemostasis in non-selected outpatients with venous thromboembolic disease was under 10%. This cast doubts on the of cost effectiveness and clinical significance of assaying hemostasis inhibitors in all such patients. The goal of this study is to evaluate the prevalence of inherited and acquired abnormalities of hemostasis in younger symptomatic outpatients with objectively diagnosed venous thromboembolic disease (VTD). METHODS: From October 1994 to October 1996, we diagnosed, treated and followed 191 consecutive outpatients with an objective diagnosis of venous thromboembolic disease, and assayed natural and acquired hemostasis inhibitors in 81 of them aged less than 50; in addition, 129 relatives of patients with inherited deficiencies were evaluated. RESULTS: Twenty-six of the patients under age 50 showed inherited deficiencies of natural inhibitors (3 antithrombin, 5 protein C, 3 protein 5 and 14 APC-R, 1 dysfibrinogenemia) and 8 patients had lupus anticoagulant (LA): abnormalities of hemostasis were found in 41.9% (95% confidence interval 31.1-53.5). In older selected patients, 60% (95% confidence interval 40.6-77.3) of the subjects showed abnormalities. Seventy-two of the relatives displayed natural inhibitor deficiencies; 88.5% of the families studied had at least one relative with the same defect as the propositus. INTERPRETATION AND CONCLUSIONS: A simple selection based on age, clinical and family history shows the existence of a high prevalence and the important clinical significance of abnormalities of hemostasis in symptomatic outpatients with venous thromboembolic disease.

Lupus anticoagulant and thrombosis: role of von Willebrand factor multimeric forms.

OBJECTIVE: Patients with lupus anticoagulant (LA) have an increased incidence of venous and arterial thrombosis whose pathogenesis is still unclear. High molecular weight von Willebrand Factor (vWF) multimers seem to play a causal role in shear stress-induced platelet aggregation and thrombus formation. We studied whether in patients with LA, alterations in the vWF multimers might coexist. METHODS: The multimeric composition of plasma vWF was analysed by SDS-electrophoresis and immunoblotting in 43 subjects positive for LA. About 2/3 of the patients had had either ischemic stroke, recurrent abortions, deep vein thrombosis (DVT) or a combination of these; the remaining subjects had never had any thrombotic events. RESULTS: An abnormal vWf multimeric pattern was found in 16 patients (37.2%); no correlation was found with the diagnosis, but the presence of abnormal vWF significantly correlated with the site of the thrombosis: indeed, it was never detected in subjects with DVT, but was found in 71.4% of patients with multiple abortions, in 50% of those with stroke and even in 25% of non-thrombotic patients. CONCLUSION: The hypothesis is put forward that abnormal VWF may represent an additional risk factor to LA for arterial thrombosis.

Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1-deamino-8-D-arginine vasopressin in humans.

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.

Factor V Arg506-->Gln mutation in patients with antiphospholipid antibodies.

Antiphospholipid antibodies (APA) have thought to be implicated in the pathogenesis of both arterial and venous thrombosis. Because of heterogeneity of APA, direct evidence of their involvement in a thrombotic event is not yet available. Development of thrombosis in the antiphospholipid antibody syndrome (APS) may occur because of the presence of additional risk factors. Here we have analysed 60 patients with APA for the presence of the Arg506-->Gln mutation in factor V. Among them 26 suffered from deep venous thrombosis, 13 from arterial thrombosis and 21 had no history of arterial or venous thrombosis. In the first group four patients were found to be heterozygous and one homozygous for the factor V Arg506-->Gln mutation. None of the patients with the factor V mutation was found in the second and third group. The incidence of factor V mutation was significantly elevated in the group of patients with venous thrombosis. These data suggest that in patients with antiphospholipid antibodies the factor V Arg506-->Gln mutation may play a major role in the occurrence of venous thrombosis.

Arterial and venous thrombosis in two Italian families with the factor V Arg506-->Gln mutation.

APC resistance, due to a point mutation in factor V at amino acid position Arg506, has been identified as a major cause of inherited thrombophilia. Here we report the presence of the factor V Arg506-->Gln mutation in 2 Italian families. In 1 family 3 subjects heterozygous and 2 subjects homozygous for the factor V Arg506-->Gln mutation were identified. The only subject who developed a thrombotic event was a 20-yr-old girl who was found to be homozygous for the factor V Arg506-->Gln mutation. In the second family 10 subjects were identified to be heterozygous for the factor V Arg506-->Gln mutation; among them 2 developed a thrombotic event. In the same family 2 individuals were found to be homozygous for the mutation: the first had a myocardial infarction at age 25 yr and the second suffered from multiple episodes of deep venous thrombosis and had a stroke at age 24 yr. These data show that the risk of developing deep venous thrombosis for the carriers of the factor V Arg506-->Gln mutation is high in the families investigated. Furthermore our data imply that the factor V Arg506-->Gln mutation in its homozygous form may relate to myocardial infarction and stroke.

[Anomalies of coagulation and fibrinolytic activity in patients with chronic pulmonary thromboembolism]. / Anomalie della coagulazione e dell'attività fibrinolitica nei pazienti con tromboembolia polmonare cronica.

INTRODUCTION: Haemostatic and fibrinolytic parameters were evaluated in patients with chronic pulmonary hypertension following pulmonary embolism (CPE). The diagnosis of CPE was based on lobar or segmental defects on perfusion lung scans and by pulmonary angiograms which showed complete or partial obstruction of main or segmentary lobar arteries. METHODS: Antithrombin III (AT III), protein C, protein S, and lupus anticoagulant (LA) were assayed in 8 patients with CPE; in 6 out of 8 patients plasma fibrinolytic activity was assessed both under basal conditions and after venous stasis. The control group consisted of 4 normal subjects. Protein C and protein S antigens were assayed by an electrophoretic method. Protein C and protein S biological activities were assayed by a manual clotting system. AT III was assayed by chromogenic method. Fibrinolytic total activity was studied on fibrin plates, tPA and PAI-1 activities by chromogenic method; tPA and PAI-1 antigens by ELISA technique. RESULTS: One patient out of 8 showed a protein C deficiency and 3 patients out of 8 were positive for a LA. All patients had a statistically significant reduction of plasma fibrinolytic activity (p < 0.001) and of tPA activity (p < 0.0005) after venous stasis as compared to the control group. CONCLUSIONS: Our data show that significant haemostatic abnormalities may underlie this disease. In particular, a) an impairment of fibrinolytic plasma activity and low levels of plasminogen activator may be found, and b) the undiagnosed presence of a LA may be the cause of these thrombotic events. The meaning of these results needs further assessment.

Lupus anticoagulant: interference with in vivo prostaglandin production and with platelet sensitivity to prostacyclin.

The hypothesis has been made that inhibition of prostacyclin (PG12) production may play a role in the pathogenesis of thrombosis in patients with the lupus anticoagulant (LA), but so far no evidence of reduced PG12 levels in vivo has been produced. We have tested the plasma levels of PG12 and thromboxane A2 (TXA2) and the platelet sensitivity to PG12 in 14 patients with and without LA and in 14 healthy controls. No significant difference in the prostanoid basal levels was detected among the groups; however, in some patients PG12 increments seemed to parallel the clinical course of the disease. Platelet sensitivity to exogenous PG12 was significantly enhanced in the LA + patients and correlated with PG12 values. We suggest that in these subjects additional factors, other than reduced PG12, may predispose to thrombosis.