The haemostatic system in acromegaly: a single-centre case-control study.

PURPOSE: Although the mortality from acromegaly is due in most cases to an increased cardiovascular risk, no study has globally evaluated the haemostatic balance in acromegaly to ascertain the presence of hypercoagulability. We endeavoured to assess the overall coagulation profile in patients with acromegaly using both traditional and global coagulation assays. METHODS: Consecutive outpatients with a diagnosis of acromegaly were enrolled and matched with healthy subjects. Whole blood thromboelastometry and impedance aggregometry, procoagulant, anticoagulant and fibrinolytic factors, as well as thrombin-generation assay and circulating endothelium-derived microvesicles were measured. RESULTS: Forty patients (M/F 14/26, median age 59 years) with either new diagnosis (naïve, 14 cases) or treated acromegaly (26 cases) were enrolled in this study. Median time from diagnosis was 11 years. Levels of factor VIII and fibrinogen were significantly higher in acromegalic patients vs. controls (p = 0.029 and < 0.003, respectively). Overall, thromboelastometry parameters showed a faster coagulation formation with a more stable clot. Acromegaly patients showed significantly higher endogenous thrombin potential [ETP] and thrombin peak compared to controls (p = 0.016 and p < 0.001, respectively). ETP remained significantly higher (p < 0.001) when thrombomodulin was added. Endothelial-derived microvesicles were significantly higher in acromegaly patients than controls (52 [40.5-67] MVs/µL and 30 [18-80] MVs/µL, p = 0.03). Patients with untreated (naïve) acromegaly showed significantly reduced ETP with and without thrombomodulin vs. patients with treated acromegaly (p = 0.01). CONCLUSION: Hypercoagulability in acromegaly is mainly due to higher levels of fibrinogen, factor VIII and thrombin generation, and appears to be more linked to the chronic phase of the disease.

"In vitro" correction of the severe factor V deficiency-related coagulopathy by a novel plasma-derived factor V concentrate.

INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.

Circulating microparticles in carriers of prothrombin G20210A mutation.

Factor V Leiden (FVL) and prothrombin gene mutation G20210A (PTM) are the two most common genetic polymorphisms known to predispose carriers to venous thromboembolism (VTE). A recent study in FVL carriers showed that circulating levels of microparticles (MP) may contribute to their thrombogenic profile. To further elucidate the prothrombotic state linked to genetic thrombophilia, we extended this study to carriers of PTM. The plasma level of annexin V-MP, endothelial-MP (EMP), platelet-MP (PMP), tissue factor-bearing MP (TF+) and the MP procoagulant activity (PPL) was measured in 124 carriers of PTM (105 heterozygous and 19 homozygous) and in 120 age- and gender-matched healthy individuals. Heterozygous and homozygous carriers of PTM showed significantly increased levels of annexin V-MP (2930 [1440-4646] MP/µl and 3064 [2412-4906] MP/µl, respectively) and significantly shorter PPL clotting time (54 [46-67] sec and 55 [46-64] sec) compared to controls (1728 [782-2122] MP/µl and 71 [61-75] sec, respectively; p<0.01). Similarly, heterozygous and homozygous subjects presented with significantly higher levels of EMP, PMP and TF+ than controls (p<0.05). PTM carriers with a VTE history had significantly higher MP numbers and activity than controls. No significant difference was seen between carriers with and without a VTE history. We conclude that the higher levels of circulating MP found in PTM carriers may play a role in the development of VTE possibly by increasing thrombin generation. Further studies are needed to better define the role of MP as triggering factors for the thrombotic complications characterizing mild genetic thrombophilic defects.

Thrombin generation in Cushing's Syndrome: do the conventional clotting indices tell the whole truth?

Cushing's Syndrome (CS) is associated with an increased mortality, where hypercoagulability seems to have a crucial role in both arterial and venous thrombosis. Parameters of in vitro thrombin generation (TG) such as lag time, peak thrombin and endogenous thrombin potential (ETP), that describe the time until thrombin burst, the peak amount of TG and the total amount of thrombin generated, respectively as well as classical clotting markers were evaluated in 33 CS patients compared to both a group of 28 patients matched for the features of Metabolic Syndrome (MetS) and 31 healthy individuals. CS and MetS patients had shorter lag time (p < 0.0001), higher peak and ETP (p < 0.0001) than healthy controls, though lag time was less shortened in CS (p < 0.0001) respect to MetS group. Prothrombin time (PT) was increased (p < 0.0001) in both CS and MetS patients, while partial thromboplastin time (PTT) was shorter (p < 0.0001) in CS compared to both MetS and healthy group (p < 0.0001). Factor VIII (FVIII), Antithrombin (AT), protein C and S were increased only in CS patients (p < 0.0001). lag time, AT and FVIII correlated to night salivary cortisol (r = + 0.59; p = 0.0005, r = + 0.40; p = 0.003, r = + 0.40; p = 0.04, respectively); PTT correlated inversely to urinary free cortisol (r = -0.45; p = 0.009). BMI correlated negatively to lag time (r = -0.40; p = 0.0001) and positively to peak and ETP (r = + 0.34; p = 0.001, r = + 0.28; p = 0.008, respectively). Obese and diabetic patients had shorter lag time (p = 0.0005; p = 0.0002, respectively), higher ETP (p = 0.0006; p = 0.007, respectively) and peak (p = 0.0003; p = 0.0005, respectively) as well as a more prolonged PT (p = 0.04; p = 0.009, respectively). Hypertensive individuals had higher ETP (p = 0.004), peak (p = 0.0008) and FVIII (p = 0.001). Our findings confirm a prothrombotic state in both CS and MetS patients, though lag time was less shortened in CS. The high levels of endogenous physiological anticoagulants, could possibly represent a protective mechanism against hypercoagulability seen in CS patients.

Factor VIIa-antithrombin complexes in patients with non-neoplastic portal vein thrombosis with and without cirrhosis.

INTRODUCTION: Portal vein thrombosis (PVT) is caused by local and systemic prothrombotic risk factors. In this case-control study, we evaluated the use of the Factor VIIa-antithrombin complex (FVIIa-AT) complex assay as a hypercoagulability marker in patients with PVT. METHODS: Two different groups of cases were considered: (i) n = 12 noncirrhotic PVT patients, (ii) n = 33 cirrhotic patients with PVT. Controls were sex and age-matched healthy volunteers and cirrhotic subjects without PVT, respectively. RESULTS: Levels of the FVIIa-AT complex were significantly higher in noncirrhotic PVT subjects (132 ± 32 pM) than in healthy volunteers (108 ± 18 pM, P = 0.04). No significant difference in FVIIa-AT complexes was seen between cirrhotic patients with (64 ± 20 pM) or without (61 ± 24 pM) PVT. A linear correlation was seen between FVIIa-AT and FVIIa in noncirrhotic PVT subjects. In cirrhotic patients, FVIIa-AT complexes depended on both FVIIa and AT. CONCLUSION: These results confirm the utility of the FVIIa-AT assay in identifying the hypercoagulable state of noncirrhotic patients because of a previous thrombotic event.

The optimal duration of anticoagulation in patients with venous thromboembolism: how long is long enough?

The risk of recurrent venous thromboembolism (VTE) approaches 40% of all patients after 10 years of follow-up. This risk is higher in patients with permanent risk factors of thrombosis such as active cancer, prolonged immobilization from medical diseases, and antiphospholipid syndrome; in carriers of several thrombophilic abnormalities, including deficiencies of natural anticoagulants; and in patients with unprovoked presentation. Patients with permanent risk factors of thrombosis should receive indefinite anticoagulation, consisting of subtherapeutic doses of low-molecular-weight heparin in cancer patients, and oral anticoagulants in all other conditions. Patients whose VTE is triggered by major surgery or trauma should be offered three months of anticoagulation. Patients with unprovoked VTE, including carriers of thrombophilia, and those whose thrombotic event is associated with minor risk factors (such as hormonal treatment, minor injuries, long travel) should receive at least three months of anticoagulation. The decision as to go on or discontinue anticoagulation after this period should be individually tailored and balanced against the haemorrhagic risk. Post-baseline variables, such as the D-dimer determination and the ultrasound assessment of residual thrombosis can help identify those patients in whom anticoagulation can be safely discontinued. As a few emerging anti-Xa and anti-IIa compounds seem to induce fewer haemorrhagic complications than conventional anticoagulation, while preserving at least the same effectiveness, they have the potential to open new scenarios for decisions regarding the duration of anticoagulation in patients with VTE.

Whole blood rotation thromboelastometry (ROTEM(®)) in nine severe factor V deficient patients and evaluation of the role of intraplatelets factor V.

Severe factor V (FV) deficiency (parahaemophilia) is a rare congenital hemorrhagic disorder characterized by very low or undetectable plasma FV levels and bleeding phenotype ranging from mild to severe. We evaluated whole blood (WB) rotation thromboelastometry (ROTEM) in parahaemophilia patients and the contribution of intraplatelets FV, if any, to clot formation. Standard ROTEM(®) assays were performed in WB from nine parahaemophilia patients and 50 healthy controls. In addition, platelets poor plasma from one parahaemophilia patient (PPP-Pt) or normal subjects (PPP-N) was reconstituted with washed platelets obtained either from one patient with parahaemophilia (Plts-Pt) or normal subjects (Plts-N) and ROTEM assays were performed in platelets rich plasma (PRP) samples. There was a prolongation of the WB clotting time (CT) in all assays in patients as compared with controls. However, maximum clot firmness (MCF) was similar in patients and controls. ROTEM in PPP-Pt showed both a prolongation of CT and a reduction of MCF as compared with PPP-N. The addition of either Plts-Pt or Plts-N to PPP-Pt resulted in similar increase in MCF and a decrease of CT which was more evident for PPP-Pt + Plts-N than PPP-Pt + Plts-Pt. In contrast, the addition of Plts-Pt or Plts-N to PPP-N had superimposable effects on both CT and MCF. In parahaemophilia patients, WB ROTEM(®) presents mainly with prolongation of CT and no relevant effect on MCF. Residual intraplatelets FV in parahaemophilia contributes significantly to thrombin generation as shown in artificially reconstituted PRP models.

Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma.

BACKGROUND: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV. OBJECTIVES: To characterize a FV-deficient patient with a severe bleeding diathesis. PATIENTS/METHODS: We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. RESULTS: The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband's plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband's whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. CONCLUSIONS: This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Prevention of thromboembolic complications in patients with atrial fibrillation.

Atrial fibrillation (AF) is a major arrhythmia in clinical practice, and its frequency rises rapidly from the sixth decade onward. Its most serious clinical consequence is ischemic stroke. Patients with AF have a five-fold increased risk of stroke compared to those in sinus rhythm. Advancing age, prior stroke or transient cerebral ischemia, diabetes, hypertension, and impaired function of the left ventricle are known risk factors. On the basis of data from several randomized controlled clinical trials and pooled analyses, several guidelines have been published to promote the use of anticoagulant treatment to prevent stroke in patients with AF. The management of oral anticoagulant therapy needs regular monitoring of INR, which should be kept in the narrow therapeutic range of 2.0-3.0 most of the time, with adjustments of the dose as required. Only a small proportion of patients with AF whom best-practice guidelines identify as eligible for oral anticoagulant therapy actually receive it. Inconvenience of monitoring and frequent dose adjustments, together with fear of major hemorrhage associated with oral anticoagulants, contribute to this underuse. In particular, conventional intensity of anticoagulation increases the risk of intracranial hemorrhage, and patients with advanced age are more prone to cerebral bleeding than younger patients. Up to date, the efficacy of aspirin, an antiplatelet agent, for stroke prevention in AF patients is less clear and remains controversial and alternative pharmacological treatment options have failed to demonstrate their superiority over vitamin K antagonists.