BACKGROUND AND OBJECTIVE:In spite of the large number of reports showing that hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis and arterial occlusive disease, this metabolite of the methionine pathway is measured in relatively few laboratories and its importance is not fully appreciated. Recent data strongly suggest that mild HHcy is also involved in the pathogenesis of venous thromboembolic disease. The aim of this paper is to analyze the most recent advances in this field. EVIDENCE AND INFORMATION SOURCES:The material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline. In addition the authors of the present article have been working in the field of mild HHcy as cause of venous thromboembolic disease. STATE OF ART AND PERSPECTIVES:The studies examined provide very strong evidence supporting the role of moderate HHcy in the development of premature and/or recurrent venous thromboembolic disease. High plasma homocysteine levels are also a risk factor for deep vein thrombosis in the general population. Folic acid fortification of food has been proposed as a major tool for reducing coronary artery disease mortality in the United States. Vitamin supplementation may also reduce recurrence of venous thromboembolic disease in patients with HHcy. At the present time, however, the clinical efficacy of this approach has not been tested. In addition, the bulk of evidence indicates that fasting total homocysteine determinations can identify up to 50% of the total population of hyperhomocysteinemic subjects. Patients with isolated methionine intolerance may benefit from vitamin B6 supplementation. Homocysteine-lowering vascular disease prevention trials are urgently needed. Such controlled studies, however, should not focus exclusively on fasting homocysteine determinations and folic acid monotherapy.

Hyperhomocysteinemia is a condition which, in the absence of kidney disease, indicates a disrupted sulfur amino acid metabolism, either because of vitamin (folate, B12 and B6) deficiency or a genetic defect. Epidemiological 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 2 to 13. This association was enhanced by including a methionine loading test. There is some evidence which suggests that hyperhomocysteinemia and APC resistance have a synergistic effect on the onset of thrombotic disease. Studies on the mechanism that underlies the relationship between thrombosis and hyperhomocysteinemia used non-physiologically high levels of homocysteine, rendering the data doubtful as to their patho-physiological relevance.

SummaryThe association of cigarette smoking with the development of occlusive vascular disease is firmly established. Unfavourable changes in a series of variables held independent risk factors for the development of vascular lesions (HDL-cholesterol, haematocrit, white blood cell count, fibrinogen and plasminogen activator inhibitor-1 (PAI-1)) are thought to be directly influenced by cigarette smoking. However, the role played by the genotype in the effect of smoking on the above parameters has not been investigated. To control the genotype, we studied the relationship between cigarette smoking and a series of cardiovascular risk factors in 27 monozygotic twin pairs (7 male and 20 female pairs, mean age ± SD: 47.4 ± 12.9 yrs) with a life-long discordance for smoking. Smoking twins had a life-long dose of smoking (Brickman index) of 287.3 ± 241.5. Body mass index, blood pressure, haematocrit, haemoglobin and red blood cell counts, total cholesterol levels and the acute phase reactants α-acid glycoprotein and C-reactive protein were similar in smokers and non-smokers. Triglyceride was higher by 12.6% (9.5-35%, 95% confidence interval, p = 0.02) and HDL-cholesterol lower by 7.5% (0.2-15%, p = 0.04) in the smoking co-twins, who also had 8.4 % (-0.2-17%, p = 0.06) higher white blood cell counts and 4.1% (1.2-7%, p <0.01) larger mean platelet volume. There was no significant difference in clottable fibrinogen (by two methods) or in the activity of plasminogen activator inhibitor-1 between the two groups, nor was the within-pair difference in these parameters related to the smoking dose. Echo-doppler examination of the carotid arteries of 24 twin pairs showed mostly minor atherosclerotic lesions in 46% and 42% of the smoking and non-smoking co-twins. After adjustment for age, systolic blood pressure and platelet count and volume were the only variables significantly associated to the presence of vascular lesions. Cigarette smoking is associated with an atherogenic lipid profile and with changes in platelets and white cells potentially reflecting endothelial cell damage. When controlling the genotype, fibrinogen and PAI-1 activity levels did not seem directly influenced by cigarette smoking.

TO THE EDITOR: Fermo and colleagues [1] clearly showed the importance of determining postmethionine loading (PML) levels of total homocysteine as a way to assess the risk for premature thromboembolic disease attributable to moderate hyperhomocysteinemia. Unfortunately, they did not measure plasma levels of pyridoxal 5-phosphate (the active metabolic form of vitamin B6). Pyridoxal 5-phosphate is the co-factor for cystathionine synthase, the key enzyme responsible for the irreversible trans-sulfuration of homocysteine. Furthermore, the presentation of the PML total homocysteine levels as absolute values (rather than as the net increase above fasting levels [2]) blurs the distinction between isolated re-methylation defects (inadequate folate or B12 status or inborn errors of folate or B12 metabolism), which result in fasting hyperhomocysteinemia with a normal increase in total homocysteine levels and isolated trans-sulfuration defects (inadequate B6 status or heterozygous cystathionine synthase deficiency), which result in essentially normal fasting total homocysteine levels with an abnormally large PML increase in total homocysteine levels [3, 4]. Without determining PLP levels, the authors cannot infer that inherited cystathionine synthase deficiency was one of the main biochemical abnormalities associated with hyperhomocysteinemia when plasma folate or B12 levels were normal. Even the control frequency of PML hyperhomocysteinemia (5%) greatly exceeds the maximum population frequency for heterozygous cystathionine synthase deficiency. A much more likely explanation for the high prevalence (approximate 22%) of PML hyperhomocysteinemia in the patients with thromboembolic disease is inadequate B6 (in the form of pyridoxal 5-phosphate) status [2-4]. The authors should consider determining fasting PLP levels in cryopreserved aliquots and reevaluating the PML findings using the net increase above fasting total homocysteine levels. These data are crucial in light of the growing momentum for homocysteine-lowering, vascular disease prevention trials that focus exclusively and inappropriately on determining fasting total homocysteine levels and folic acid monotherapy [5].

Patients with antiphospholipid antibody syndrome (APS) experience a higher rate of recurrence of thrombosis than the general population of patients with thrombotic disease. Based on a retrospective analysis, it has been suggested that patients with APS should be kept on prolonged anticoagulation aiming at international normalised ratio (INR) values > 3.0. To evaluate whether the requirement for more intense anticoagulation depends on the variable sensitivity of thromboplastin reagents to the influence of aPLA, we monitored oral anticoagulant treatment in 10 patients with persistent lupus anticoagulants (LA) and venous thromboembolic disease using two thromboplastin reagents: Pro-IL-Complex (Instrumentation Laboratory, combined) and Recombiplastin (Ortho, recombinant). Acenocoumarol dosage was always assigned based on INR values obtained with the combined thromboplastin using diluted (1:20) test plasma, aiming at an INR interval of 2.0 to 3.0. Single INR determinations with both reagents were obtained throughout the study period for 110 aPLA-free patients on stable oral anticoagulation. Using the manufacturer's instrument-certified international sensitivity index (ISI) values, INR obtained with the recombinant reagent were significantly higher than those obtained with the combined reagent in LA-positive patients, but they were lower in LA-negative patients. After correction for local ISI calibration in LA-negative patients, INR values of 3.1 and 4.6 with Recombiplastin corresponded, respectively, to INR values of 2.0 and 3.0 with Pro-IL-Complex. These results indicate the thromboplastin-dependency of INR values in patients with LA, thereby questioning the validity of the INR system for the monitoring of oral anticoagulant treatment in these patients.

The major improvements obtained in the quality of commercial reagents and in the accuracy of coagulometers has rendered comparabilty of the results among different laboratories a major task of the bodies devoted to standardization of coagulation testing. Commutability of results is of major importance especially in the monitoring of anticoagulant therapy. Critical aspects relate to the preanalytical phase, the anaytical phase and to the expression of results with the adoption of proper correction factors - like the international sensitivity index (ISI) for thromboplastin reagents. Problems with the APTT in the monitoring of heparin treatment include the variable sensitivity of commercially available APTT reagents to clinically insignificant deficiencies of factors involved in the contact phase of coagulation and to the presence of lupus anticoagulants, which - in addition to the different sensitivity of the reagents to the anticoagulant effect of heparin - render commutability of results unworkable in practice.

SummaryTo determine their ability to diagnose postoperative deep vein thrombosis (DVT) D-dimer – by three methods – fibrinogen degradation products (FgDP) and fibrinogen levels were measured in 68 consecutive patients before elective surgery for hip replacement and on postoperative day 1, 3, 6, and 10. All patients received prophylaxis and underwent compression real-time B-mode ultrasonography (C-US) on postoperative day 5 and 9, and bilateral ascending venography on day 10. Twenty-two out of 68 patients developed asymptomatic postoperative DVT, which was limited to the calf veins in 14 and involved the proximal veins in 8 patients. C-US was negative in all patients on day 5. On day 9, C-US sensitivity and specificity for proximal DVT were 63% (95% confidence interval: 26%-90%) and 98% (89%-100%) respectively. Postoperative changes in the laboratory parameters evaluated were not different in patients with or without DVT until day 10. On day 10, mean D-dimer, FgDP and fibrinogen levels were significantly higher in patients with DVT than in those without DVT (p values between 0.006 and 0.032), but only D-dimer was higher with DVT involving two or more venous segments than with thrombosis involving one venous segment only (p <0.05). Stepwise logistic regression analysis identified D-dimer and fibrinogen on day 10 as predictors of postoperative DVT. In a receiver operator curve and after weighing for the coefficients generated by logistic regression analysis, the combination of a latex photometric immunoassay and of PT-derived fibrinogen yielded - at a cut-off value of 7.0 - a sensitivity of 100% (73%-100%) and a specificity of 58% (39%-75%) for DVT, with a negative predictive value of 100% (78%-100%), a positive predictive value of 52% (32%-71%) and an overall accuracy of 71% (55%-83%). These results suggest that two simple, fast and reproducible tests may permit the identification of patients at low risk of having postoperative DVT and that a combination of sensitive laboratory assays and of the highly specific C-US may select patients requiring anticoagulant treatment. Efficacy and cost-effectiveness of this approach should be evaluated in large clinical management studies.

We describe a case of central retinal vein and branch artery occlusion associated with inherited type I plasminogen deficiency (68%) and permanent elevation of Lp(a) (460 mg/l, S-2 phenotype) in a 45 year old white woman with no associated local or systemic risk. factors. Pedigree analysis revealed inheritance of plasminogen deficiency from the deceased father and of high Lp(a) levels from the mother. Both the patient's sons had plasminogen deficiency, but they had normal Lp(a) levels. In a series of 40 consecutive patients with central retinal vein occlusion we previously reported the observation of high Lp(a) levels - consistently associated with the S-2 phenotype - in 30% of the patients as compared to a 10% incidence in controls. This case emphasizes the importance of screening patients with occlusion of the retinal vessels and no associated risk factors for coagulation abnormalities predisposing to thrombosis.

Congenital and acquired deficiencies of vitamin K-dependent protein S (PS) are an established risk factor for thrombotic disease (1,2). Due to the multiple PS domains involved in the interaction with other plasma proteins and the expression of activated protein C (APC) cofactor activity, the existence of qualitative defects of PS is anticipated (3). Thus, measurement of PS anticoagulant activity should be preferred to PS antigens determination in the identification of both congenital and acquired deficiencies. At present, two commercial assays are available for the measurement of the APC cofactor activity of PS. with one of these assays qualitative protein S deficiencies had been erroneously identified (4), as originally indicated by a multicenter collaborative study comparing different functional PS assays (5). In the latter study, plasma samples obtained from patients reportedly affected by qualitative PS deficiencies showed virtual coincidence of free PS antigen and anticoagulant activity levels only when this was evaluated after immunoadsorption of PS (6). This observation raised the possibility of artifacts related to PS testing in whole plasma (5). Following the description of APC resistance a new marker of congenital thrombophilia characterized by defective clotting time prolongation upon additon of APC to plasma (7) -, the seemingly PS deficient patients have been correctly diagnosed as APC resistant cases, with the conclusion that APC resistance may interfere in PS activity testing by the available commercial methods (4). To explore the extent of such interference, we have conducted a prospective comparison of two commercial functional PS assays and our home-made assay (6) in a series of patients with APC resistance referred to our Institution for the evaluation of a thrombophilic state.

Poor comparability of two commercial ELISA methods for the measurement of prothrombin fragment 1.2 (FI.2) was recently reported (1). Because of the potential utility of plasma F1.2 measurement in the monitoring of the in vivo action of oral anticoagulant drugs (2), we con­ ducted a study aimed to evaluate the comparability of two commercial kits for the measurement of F 1.2 levels in normal controls (n = 80, 40 men and 40 females, mean age 40.8 ± 12.2 yrs, range 18-69) and in patients on stabilized oral anticoagulant treatment (n = 99, mean INR 2.62 ± 1.07, range 1.20-8.25). Blood was drawn sequentially - using a 20 gauge needle - into 5 ml vacutainer tubes (Becton Dickinson) con­ taining 0.129 M sodium citrate or sodium heparin. Plasma F1.2 levels were tested with the Enzygnost FI+2 (Behring, Marburg, Germany) in citrated plasma and with the FI.2 ELISA (Baxter, Miami, FI) in both citrated and heparinised plasma. The manufacturer’s instructions were followed closely except for omitting the 10 nM calibrator and adding a 1 nM calibrator - obtained by dilution of the 2nM calibrator in Tris-buffered saline - in the Behring calibration curve. In spite of the different mean values (Baxter-heparin 0.26 ±0.15 nM, Baxter-citrate 0.17 ± 0.10 nM, Behring-citrate 0.64 ± 0.29 nM) and of the moderate correlation observed between the values determined with the Behring-citrate and the Baxter-heparin methods (r = 0.712), there were good correlations between the FI.2 values