Armando D'Angelo

Bleeding complications of oral anticoagulant treatment: an inception-cohort, prospective collaborative study (ISCOAT)

BACKGROUND Bleeding is the most serious complication of the use of oral anticoagulation in the prevention and treatment of thromoboembolic complications. We studied the frequency of bleeding complications in outpatients treated routinely in anticoagulation clinics. METHODS In a prospective cohort from thirty-four Italian anticoagulation clinics, 2745 consecutive patients were studied from the start of their oral anticoagulation (warfarin in 64%, acenocourmarol in the rest). The target anticoagulation-intensity was low (international normalised ratio [INR] < or = 2.8) in 71% of the patients and high (> 2.8) in the remainder. We recorded demographic details and the main indication for treatment and, every 3-4 months, INR and outcome events. Such events included all complications (bleeding, thrombosis, other), although only bleeding events are reported here, and deaths. We divided bleeding into major and minor categories. FINDINGS 43% of the patients were women. Nearly three-fifths of the patients were aged 60-79; 8% were over 80. The main indication for treatment was venous thrombolism (33%), followed by non-ischaemic heart disease (17%). Mean follow-up was 267 days. Over 2011 patient-years of follow-up, 153 bleeding complications occurred (7.6 per 100 patient-years). 5 were fatal (all cerebral haemorrhages, 0.25 per 100 patient-years), 23 were major (1.1), and 125 were minor (6.2). The rate of events was similar between sexes, coumarin type, size of enrolling centre, and target INR. The rate was higher in older patients: 10.5 per 100 patient-years in those aged 70 or over, 6.0 in those aged under 70 (relative risk 1.75, 95% Cl 1.29-2.39, p < 0.001). The rate was also higher when the indication was peripheral and/or cerebrovascular disease than venous thromboembolism plus other indications (12.5 vs 6.0 per 100 patient-years) (1.80, 1.2-2.7, p < 0.01), and during the first 90 days of treatment compared with later (11.0 vs 6.3, 1.75, 1.27-2.44, p < 0.001). A fifth of the bleeding events occurred at low anticoagulation intensity (INR < 2, rate 7.7 per 100 patient-years of follow-up). The rates were 4.8, 9.5, 40.5, and 200 at INRs 2.0-2.9, 3-4.4, 4.5-6.9, and over 7, respectively (relative risks for INR > 4.5, 7.91, 5.44-11.5, p < 0.0001). INTERPRETATION We saw fewer bleeding events than those recorded in other observational and experimental studies. Oral anticoagulation has become safer in recent years, especially if monitored in anticoagulation clinics. Caution is required in elderly patients and anticoagulation intensity should be closely monitored to reduce periods of overdosing.

Enoxaparin plus compression stockings compared with compression stockings alone in the prevention of venous thromboembolism after elective neurosurgery.

Background Compression stockings are recommended for prophylaxis against venous thromboembolism in patients undergoing neurosurgery, but anticoagulant agents have not gained wide acceptance because of concern about intracranial bleeding. Methods In a multicenter, randomized, double-blind trial, we assessed the efficacy and safety of enoxaparin in conjunction with the use of compression stockings in the prevention of venous thromboembolism in patients undergoing elective neurosurgery. Enoxaparin (40 mg once daily) or placebo was given subcutaneously for not less than seven days beginning within 24 hours after surgery. The primary end point was symptomatic, objectively confirmed venous thromboembolism or deep-vein thrombosis assessed by bilateral venography, which was performed in all patients on day 8±1. Bleeding side effects were carefully assessed. Results Among the 307 patients assigned to treatment groups, 129 of the 154 patients receiving placebo (84 percent) and 130 of the 153 patients receiving enoxaparin (85 percent) had venographic studies adequate for analysis. An additional patient in the placebo group died before venography of autopsyconfirmed pulmonary embolism. In this analysis, 42 patients given placebo (32 percent) and 22 patients given enoxaparin (17 percent) had deep-vein thrombosis (relative risk in the enoxaparin group, 0.52; 95 percent confidence interval, 0.33 to 0.82; P=0.004). The rates of proximal deep-vein thrombosis were 13 percent in patients receiving placebo and 5 percent in patients receiving enoxaparin (relative risk in the enoxaparin group, 0.41; 95 percent confidence interval, 0.17 to 0.95; P=0.04). Two patients in the placebo group died of autopsy-confirmed pulmonary embolism on days 9 and 16. Major bleeding occurred in four patients receiving placebo (intracranial bleeding in all four) and four patients (intracranial bleeding in three) receiving enoxaparin (3 percent of each group). Conclusions Enoxaparin combined with compression stockings is more effective than compression stockings alone for the prevention of venous thromboembolism after elective neurosurgery and does not cause excessive bleeding. (N Engl J Med 1998; 339:80-5.)

Natural history and risk factors for thrombosis in 360 patients with antiphospholipid antibodies: A four-year prospective study from the italian registry

PURPOSE To assess the natural history and risk factors for thrombosis in a large cohort of unselected patients with antiphospholipid antibodies. PATIENTS AND METHODS Three hundred sixty consecutive patients (118 males, 242 females, median age 39 years [range 2 to 78]) fulfilling the currently accepted criteria for diagnosis of lupus anticoagulant (LAC) (n = 326) and/or raised immunoglobulin G anticardiolipin antibodies (IgG ACA) (n = 185) were collected from 16 Italian institutions and prospectively observed for a median of 3.9 years (range 0.5 to 5). Main endpoints were the occurrence of arterial or venous thrombosis, the outcome of pregnancies, and any severe complications leading to hospitalization or death. RESULTS Thirty-four patients developed a thrombotic complication, with a total incidence of 2.5% patient-years. Multivariate logistic regression analysis identified two independent risk factors for thrombotic events: a previous thrombosis (RR 4.9; 95% CI, 1.76 to 13.7; P < 0.005) and IgG ACA titer above 40 units (RR 3.66; 95% CI, 1.24 to 10.8; P < 0.01). A total of 28 pregnancies were observed in 25 women and 11 (39%) were abortive. Adverse pregnancy outcomes were significantly more frequent in women with a history of miscarriage or vascular occlusion (9/16, 56%) than in asymptomatic women (2/12, 17%) (P = 0.035). Four patients developed non-Hodgkins lymphoma during the follow-up. Eighteen patients died. Vascular events and hematological malignancies represented the most frequent causes of death (n = 5 for each). CONCLUSIONS The present study shows that: (a) previous thrombosis and ACA titer > 40 U are independent predictors of thrombosis; (b) history of miscarriage or vascular disease is significantly associated with adverse pregnancy outcome; (c) hematological malignancies can develop during follow-up in patients with antiphospholipid antibodies.

Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease

Homocysteine, a thiol-containing amino acid, is the branch point between the transsulfuration and the transmethylation pathways of methionine [1]. In the last decade, investigators have studied the role of homocysteine metabolism in the pathogenesis of occlusive vascular disease. Homocystinuria, a disorder caused by homozygous cystathionine -synthase (E.C. 4.2.1.22) deficiency or defects in the vitamin B12 or folate-dependent remethylation of homocysteine to methionine and characterized by fasting plasma homocysteine levels of 200 mol/L or greater, is frequently associated with severe vascular disease during infancy or childhood. More than half of homocystinuric patients have a first thromboembolic event before the age of 30 years, and premature atherosclerosis is a common development [2]. Although the relation of hyperhomocysteinemia to the occurrence of vascular disorders is still unclear, it is of increasing interest that less severe enzymatic abnormalities of the methionine metabolic pathway may predispose to the development of premature vascular disease. Moderate hyperhomocysteinemia, which is characterized by elevations of fasting plasma homocysteine levels as high as 100 mol/L or by abnormally high levels of plasma homocysteine after methionine load [3], has been observed with increasing frequency in young patients with arterial occlusive conditions such as myocardial infarction and cerebrovascular and peripheral occlusive diseases [4-7]. However, although venous thromboembolism accounts for 50% of the vascular complications of homocystinuria, few data are available on the relation between moderate hyperhomocysteinemia and venous thromboembolic disease [8, 9]. The term thrombophilia refers to an increased tendency to thrombosis sustained by an ongoing stimulus to thrombogenesis or by a defect of the natural anticoagulant or fibrinolytic mechanisms [10]. Genetic factors play an important role in thrombophilia because thrombosis is often familial or is associated with congenital deficiencies of the protein C anticoagulant pathway, antithrombin III, heparin cofactor II, or plasminogen. The main clinical features of patients with inherited thrombophilia are recurrent thrombosis, thrombosis at a young age, idiopathic thrombosis, thrombosis after trivial provocation, and thrombosis in an unusual site [10]. The aim of our study was to evaluate the prevalence of moderate hyperhomocysteinemia and of established disorders of inherited thrombophilia in a series of consecutive patients with early-onset venous or arterial occlusive disease. Methods Patients We studied consecutive unrelated patients referred to our coagulation service from November 1992 to October 1994. Patients were enrolled in the study if they were younger than 45 years at the time of the occlusive event (n = 142) or if they had thrombosis at unusual sites (central retinal vein or artery, cerebral veins), provided that local risk factors were absent. Exclusion criteria were the presence of overt cancer, liver disease, acquired coagulation abnormalities (for example, lupus anticoagulants, anti-protein S antibodies, and inhibitors of fibrin polymerization), or risk factors known to disturb methionine metabolism (diabetes, hypertension, hyperlipidemia and renal failure). Thirty-two patients referred during the study period were excluded on the basis of these criteria. At the first study visit, patients were asked [by means of a standard questionnaire] about their personal and family history of thrombosis and whether they had predisposing factors for thrombosis. Diagnoses were confirmed by objective methods as follows: 1) Peripheral arterial occlusive disease was confirmed by Doppler ultrasonography and angiography; 2) cerebrovascular disease was confirmed by angiography and computed tomography or nuclear magnetic resonance imaging; 3) arterial or venous retinal occlusion was confirmed by retinal fluorescein angiography; 4) deep venous thrombosis was confirmed by ultrasonography or phlebography; 5) caval or pelvic venous thrombosis was confirmed by color flow Doppler imaging; 6) pulmonary embolism was confirmed by scintigraphy or angiography; and 7) myocardial infarction was confirmed by electrocardiography. The control group consisted of 60 apparently healthy persons. These controls were either subjects, students, or members of the hospital staff (30 women and 30 men; mean age SD, 35.8 11.8 years) who were not taking any medications and who were recruited during the study period. Patients who were receiving antiplatelet drugs were not excluded from the investigation. All patients had blood drawn at least 3 months after the occlusive event if they were not receiving heparin or oral anticoagulant treatment. If patients were receiving anticoagulant treatment, laboratory evaluation was done 6 months after the event and at least 1 week after interruption of oral anticoagulant treatment. Therapies known to induce variations in homocysteine levels were not given to any patient before testing. All study participants gave informed consent. Laboratory Studies Venous blood samples were obtained in vacutainer tubes (Becton Dickinson, Rutherford, New Jersey) containing 0.129 M sodium citrate (9:1, v/v) from an antecubital vein and centrifuged within 30 minutes at 2000 g for 10 minutes at room temperature. Plasma aliquots were snap-frozen with methanol and dry ice and stored at 70 C. Assays were done within 3 months of blood collection. Total plasma homocysteine, which corresponds to the sum of free homocysteine, cysteine-homocysteine mixed disulfide, and protein-bound forms, was obtained after cleavage and reduction reactions with sodium borohydride followed by iodoacetic acid treatment. Homocysteine and O-phthaldialdehyde were purchased from Fluka (Buchs, Switzerland). All chemicals and solvents were obtained from BDH (Poole, United Kingdom). High-performance liquid chromatographic analysis was done using the O-phthaldialdehyde precolumn derivatization method [11]. Plasma vitamin B12 and folate levels were determined by using chemiluminometric immunoassays (Ciba Corning ACS, Medfield, Massachusetts). Because heterozygosity for cystathionine -synthase deficiency in patients who have normal fasting plasma homocysteine levels may be detected by an abnormal increment in the total plasma homocysteine level after administration of an oral methionine load [1], all patients older than 14 years were asked to receive oral methionine and have their plasma homocysteine levels measured. Eighty-seven patients (55%) gave their consent. After an overnight fast, a venous blood sample was drawn at 0800 h and L-methionine (0.1 g/kg body weight) was orally administered in 200 mL of fruit juice. Methionine intake was followed by a simple Italian coffee breakfast, and 5 hours later by a regular lunch. In a previous study [12], no diurnal variation in total plasma homocysteine levels in relation to food intake had been observed. The blood specimen was taken 8 hours after methionine intake. In 12 healthy persons, the transient increase in plasma homocysteine level peaked 6 to 8 hours after methionine administration. Increments in plasma homocysteine levels after methionine load were measured in controls. The activity of antithrombin III (Coatest Antithrombin, Chromogenix, Stockholm, Sweden) and plasminogen (Coatest Plasminogen, Chromogenix) were tested by amydolytic methods adapted to the ACL 300 (Instrumentation Laboratories, Milan, Italy). The activity of heparin cofactor II was tested as previously described [13], and protein C and protein S anticoagulant activity [14, 15] and antigens [14, 16] were measured by previously described methods. The anticoagulant response to activated protein C was tested in an activated partial thromboplastin time-based assay using a commercial reagent (Actin FSL, Baxter, Miami, Florida) and human purified protein C activated by the thrombin-thrombomodulin complex [17]. Because this assay was introduced in our laboratory in March 1993, patients who were enrolled in the study before this date were reinvestigated for activated protein C resistance. Family Studies When an abnormality in the laboratory variables was detected, a second blood sample was taken to confirm the observation. In addition, whenever possible, the relatives of the propositus were studied to confirm inheritance of the abnormality. First-degree relatives and siblings of 12 patients with mild hyperhomocysteinemia were studied both while fasting and after methionine intake. For 8 of these families, both parents of the proband were available for study. First-degree relatives and siblings of 11 probands were evaluated for activated protein C resistance and deficiency of protein S, protein C, and plasminogen. Statistical Analysis Results are expressed as mean SD or as median and range. Hyperhomocysteinemia was defined by a persistent elevation of fasting plasma homocysteine levels or by plasma homocysteine levels after methionine load that exceeded the 95th percentile of the distribution in the control group in the presence of normal folate and vitamin B12 plasma levels [5, 18]. In children, hyperhomocysteinemia was diagnosed when fasting plasma homocysteine levels were greater than the 95th percentile of an age-matched control group (12 mol/L [12]). Deficiency of protein C, protein S, and plasminogen and resistance to activated protein C were confirmed by the observation of values persistently lower than the 5th percentile distribution of values in the control group. In the control group, all laboratory variables were normally distributed except total plasma homocysteine levels, which were log-transformed to approximate normal distribution. Homocysteine levels are lower in women than in men [12, 19]. Because sex-related differences in plasma homocysteine levels were also observed in our control group (P < 0.0001), separate 95th percentiles were used for men (fasting levels, 19.5 mol/L; lev

Diagnosis and treatment of disseminated intravascular coagulation: Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET)☆

BACKGROUND The diagnosis and treatment of disseminated intravascular coagulation (DIC) remain extremely controversial. PURPOSE The Italian Society for Thrombosis and Haemostasis commissioned a project to develop clinical practice guidelines for the diagnosis and treatment of DIC. METHODS Key questions about the diagnosis and treatment of DIC were formulated by a multidisciplinary working group consisting of experts in clinical medicine and research. After a systematic review and discussion of the literature, recommendations were formulated and graded according to the supporting evidence. In the absence of evidence, evidence of low quality, or contradictory evidence, a formal consensus method was used to issue clinical recommendations. RESULTS AND CONCLUSIONS In suspected DIC, we suggest the use of the diagnostic scores ISTH (grade C), JMHW (grade C) or JAAM (grade D) over stand alone tests. The cornerstone of the management of DIC remains the treatment of the underlying triggering disease. We do not suggest the use of antithrombin (grade D), dermatan sulphate (grade D), gabexate (grade D), recombinant factor VIIa (grade D), activated protein C (grade D), thrombomodulin (grade B). The use of unfractionated heparin or low-molecular-weight heparin is not suggested except for thromboembombolic prophylaxis in patients a high risk who do not have active bleeding (grade D). In patients with severe sepsis/septic shock and DIC we suggest the use of human recombinant activated protein C (grade D). In patients with DIC and active bleeding we suggest the use of transfusion therapy (platelets, plasma, cryoprecipitate) (grade D).

Hypercoagulability in acute stroke Prognostic significance

To evaluate the clinical significance of hemostatic abnormalities in acute stroke, we studied coagulation and platelet function in 70 patients with recent cerebral infarction or hemorrhage and in 45 age-matched controls. Higher levels of one-stage factor VIII coagulant activity, fibrinopeptide A (FPA), and beta-thromboglobulin were associated with the occurrence of stroke. All hemostatic test results were remarkably similar in patients with ischemic and hemorrhagic stroke. FPA levels and size of the lesion on CT were the only variables independently predicting mortality in a multivariate regression analysis. Our findings demonstrate that hypercoagulability is an important prognostic factor in stroke and lend support to clinical trials of drugs interfering with the coagulation system in the early phase of cerebral ischemia.

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).

Relationship Between Homocysteine and Thrombotic Disease

Hyperhomocysteinemia is a condition which, in the absence of kidney disease, indicates a disrupted sulfur amino acid metabolism, either because of vitamin deficiency (folate, B12 and B6) or a genetic defect. Epidemiologic evidence suggests that mild hyperhomocysteinemia is associated with increased risk of arteriosclerotic disease and stroke. The relationship between hyperhomocysteinemia and thrombosis has been investigated in 10 studies involving a total of 1200 patients and 1200 controls. Eight of these studies demonstrated positive association with odds ratios that ranged from two to 13. This association was enhanced by including a methionine loading test. There is some evidence which suggests that hyperhomocysteinemia and activated protein C resistance have synergistic effect on the onset of thrombotic disease. Recent studies with animal models for mild hyperhomocysteinemia provided encouraging results in the understanding of the mechanism that underlies this relationship between mild elevations of plasma homocysteine and vascular disease. These animal models pointed to the possibility that the effect of elevated homocysteine is multifactorial, affecting both the vascular wall structure and the blood coagulation system.

Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.

Hypercoagulability during L-asparaginase treatment: the effect of antithrombin III supplementation in vivo.

To evaluate the occurrence of hypercoagulability during treatment with l‐asparaginase (l‐ase), thrombin‐antithrombin complex (TAT) and d‐dimer levels in plasma were serially measured in 15 consecutive adult patients with acute lymphoblastic leukaemia or lymphoblastic lymphoma who had recently completed a chemotherapy cycle with cytosine arabinoside and methotrexate. The first eight patients (group A) received i.v. l‐ase alone (20000 U/m2 on alternate days over 10 d); the last seven patients (group B) received, in addition to l‐ase, bolus injection of antithrombin concentrate (2000 U) on alternate days for a total of six administrations, beginning with the second l‐ase infusion. Increased levels of TAT (P<0·05) and d‐dimer (P<0·01) were observed prior to l‐ase, possibly related to inflammation and cytolysis secondary to previous chemotherapy. In patients treated with l‐ase alone, further elevation of TAT (P<0·05) and persistence of increased d‐dimer were observed, associated with marked reduction of the anticoagulant activities of protein C, protein S and antithrombin III. At variance, in patients receiving antithrombin III supplementation there was no increase of TAT and a normalization of d‐dimer levels occurred during l‐ase treatment. In these patients, mean plasma antithrombin III activity was maintained at levels higher than 70% of normal throughout the treatment. The rate of decline of fibrinogen, factor IX, protein C and protein S was unaffected by antithrombin III supplementation, indicating that hypercoagulability has little if any relevance for the reduction of coagulation factors and inhibitors induced by l‐ase treatment. The usefulness of antithrombin III concentrates in preventing thromboembolic complications in patients submitted to l‐ase treatment remains to be determined.