Thromb Haemost 2004; 91: 506-13form. No release of MMP-2 from the graft could be detected. In contrast, plasma levels of MMP-9 increased sharply during the anhepatic and postreperfusion periods. Peak MMP-9 levels of about eight times above baseline were found at 30 minutes after reperfusion. Most MMP-9 appeared to be in its active/inhibitorcomplexed form. No significant differences were observed between the three treatment groups. However, in patients with more severe ischemia/reperfusion (I/R) injury the MMP-9 concentration, particularly of the active/inhibitor-complexed form, remained high at 120 minutes postreperfusion compared to patients with no or mild I/R injury. The decrease in plasma levels of MMP-2,TIMP-1 and TIMP-2 during OLT occurred irrespective of the severity of the I/R injury. There was a significant correlation between MMP-9 and t-PA levels, but not with TNF-α. In conclusion, OLT is associated with a sharp increase of MMP-9 during the anhepatic and postreperfusion periods, which coincided with the changes in t-PA. MMP-2, TIMP-1 and TIMP-2 gradually decreased during OLT. The composition of these MMPs was not altered by the use of aprotinin, suggesting that serine-protease/plasmin-independent pathways are responsible for MMP regulation during OLT. In addition, only MMP-9 seems to be involved in I/R injury during human liver transplantation. KeywordsAprotinin, ischemia/reperfusion injury, liver transplantation, matrix metalloproteinases, plasmin Summary Uncontrolled activation of matrix metalloproteinases (MMPs) can result in tissue injury and inflammation, yet little is known about the activation of MMPs during orthotopic liver transplantation (OLT). OLT is associated with increased fibrinolytic activity due to elevated plasmin generation. The serine-protease plasmin not only causes degradation of fibrin clots but is also thought, amongst others, to play a role in the activation of some matrix metalloproteinases.We therefore studied the evolution of MMP-2 and -9 plasma concentrations during OLT and the effect of serine-protease inhibition by aprotinin on the level and activation of these MMPs. In a group of 24 patients who participated in a randomized, double-blind, placebo-controlled study we determined serial MMP-2 and MMP-9 plasma levels during transplantation using ELISA (total MMP), activity assays (activatable MMP) and zymography. In addition, the MMP-inhibitors TIMP-1 and TIMP-2 were assessed by ELISA. The putative regulating factors tumor necrosis factor alpha (TNF-α) and tissue-type plasminogen activator (t-PA) were assessed as well. Patients were administered high-dose aprotinin, regular-dose aprotinin or placebo during surgery. Plasma TIMP-1, TIMP-2 and MMP-2 level gradually decreased during transplantation. Approximately twothirds of total MMP-2 appeared to be in its activatable proMMP Correspondence to:

SummaryThe aim of this study was to evaluate the relationship between factor VIII (FVIII) levels, measured by chromogenic and clotting assays, and risk of venous thromboembolism (VTE) recurrence. A total of 564 patients underwent clinical follow-up after oral anticoagulant withdrawal (total follow-up ¼ 924AE4 years). Recurrent VTE developed in 39 of 309 (12AE6%) patients with a first idiopathic VTE and in 14 of 255 (5AE5%) patients whose first event was secondary. In patients with a first idiopathic VTE, the risk of recurrence was more than fivefold higher in patients with FVIII levels exceeding the 90th percentile [chromogenic FVIII: relative risk (RR) 5AE43 (95% CI 1AE76-16AE8); clotting FVIII: RR 6AE21 (95% CI 1AE57-24AE5)] after adjustment for all possible confounding variables. In patients with a first secondary VTE, the risk of recurrence was slightly higher in patients with high FVIII levels [chromogenic FVIII: RR 2AE62 (95% CI 0AE34-19AE9); clotting FVIII: RR 1AE74 (95% CI 0AE25-12AE1)], but, given the low number of recurrences, the 95% CI were very large. In conclusion, this study shows that high FVIII levels are associated with increased risk of VTE recurrence in patients with a first idiopathic VTE. Although the measurement of FVIII levels by a specific chromogenic assay might, in principle, be preferred to avoid the risk of aspecific clotting effects, no significant differences in results obtained by chromogenic or clotting methods were found.

Common inherited thrombophilic defects such as factor V Leiden and G20120A mutation of the prothrombin gene interact synergistically with oral contraceptives to increase the risk of venous thromboembolism.1 2 The best approach to identify women at higher risk of venous thromboembolism before taking oral contraceptives is controversial. Universal screening is not cost effective because 8000 women need to be screened for factor V Leiden to detect 400 mutations and prevent one episode of venous thromboembolism.1 Many authors recommend selective screening in women with a personal or family history of venous thromboembolism. 1 However, the effectiveness of this approach has not been proved. The aim of our study was to evaluate the sensitivity and positive predictive value of a family history of venous thromboembolism for identifying common thrombophilic defects in women without thrombosis before taking oral contraceptives. Participants, methods, and resultsWe prospectively evaluated a cohort of women (age range 15-49 years) consecutively referred to our thrombophilia unit by gynaecologists at family planning clinics in Bologna, Italy, between 1998 and 2000. The gynaecologists had established that the women were eligible to take oral contraceptives and had no history of venous thromboembolism. Before the women were screened, experienced investigators administered a modified structured questionnaire 3 that was designed and validated to evaluate both personal and family history (first degree = parents and siblings, second degree = grandparents, aunts, uncles, and cousins) of venous thromboembolism (see BMJ's website for details). We considered family history positive if a thromboembolism was reported in any first or second degree relatives.Thrombophilia screening was conducted as previously described. 4 Prothrombin activity was measured by chromogenic assay 5 and lupus anticoagulant by LA-test and LA-check assays (Organon Teknika, Rome, Italy). If prothrombin activity was confirmed to be above 1.10 U/ml, we analysed the DNA for the G20120A mutation according to the method of Poort et al. 5 The tests were performed by staff blind to the results of the questionnaire.We calculated sensitivity and positive predictive values according to standard methods. The 95% confidence intervals for proportions were calculated by an approximate method, and we used the 2 test when appropriate. A two sided probability value < 0.05 was considered significant. All data were analysed with the statistical package SOLO ( BMDP, Los Angeles).We evaluated 324 women (mean age 34 years) who had a negative personal history for venous thromboembolism confirmed by our questionnaire. Thirty four women reported a positive family history (10%, 95% confidence interval 7% to 14%), of whom two were heterozygous for factor V Leiden and one had protein S deficiency. Thrombophilic defects were identified in 19 women (6%, 3% to 8%), only three of whom had a positive family history. Among the 290 women with a negative family history, thrombophilic defects w...

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Summary305 patients with juvenile thromboembolic episodes were screened for the presence of heparin cofactor II deficiency. The heterozygous deletion of two bases was found in the exon 5 of the heparin cofactor II gene in two unrelated patients, very likely due to a founder effect. This molecular lesion, causing a frameshift and elongated translation, affects the core of the molecule and should cause the complete unfolding of the protein, which is in accordance with the observed type I deficiency. The corresponding region of antithrombin III gene is affected by a cluster of frameshift mutations suggesting that heparin cofactor II and antithrombin III could share similar mutational patterns.The heparin cofactor II gene alteration was associated with, in one patient, the factor V Leiden mutation and, in the other, type I protein C deficiency. The tracing of the single defects in several family members indicated that the mutations became clinically manifest only when present in the doubly heterozygous condition. This study provides two examples, based on molecular findings, of the interplay of risk factors which is potentially useful to define a role for heparin cofactor II deficiency in inherited thrombophilia.