The clinical phenotype of patients with haemophilia A (HA) often differs between individuals with the same factor VIII (FVIII) gene defect (e.g. within the same family) or the same coagulant activity of FVIII (FVIII:C). We proposed that because the thrombin generation assay in platelet-poor plasma of HA patients provides more information [peak thrombin concentration, endogenous thrombin potential (ETP), rate of thrombin generation and lag-time] than a clot-based FVIII assay it might provide insight into these differences. We therefore investigated the relation between the results of the thrombin generation assay and the clinical severity in nine families with HA (23 patients with different phenotypes). We also examined the contribution of prothrombotic risk factors: (FV Leiden G1691A and prothrombin G20210A), the coagulant activity of FVIII and tissue factor (5'UTR) polymorphisms. Our data detect marked differences between individuals but these did not correlate with the reported clinical phenotype. These differences were also reflected in a marked difference in response to the therapeutic amounts of FVIII. This might account for differences in amounts of treatment consumption. Reduced peak and possibly rate of thrombin generation, rather than FVIII:C or ETP appear to represent the critical defects in FVIII-deficient plasma. We suggest that the analysis of parameters in thrombin generation is a useful tool to detect bleeding tendency in HA but not to predict the modulation of the haemorrhagic tendency in patients within families. However the presence of the other factors such as vessel wall components, protein C and platelets might need to be incorporated into this system.

Hemophilia A (HA) is one of the most common inherited bleeding disorders caused by FVIII gene mutations. Inversion of intron 22 (inv22) originates 50% of cases of severe HA and is a major risk factor for inhibitor development. Inversion of intron 1 (inv1) has been reported to occur in 2-3% of severe HA patients. We studied both inversions to determine their frequencies in Mexican patients with severe HA and to compare these data with other HA populations. The inv22 was evaluated as a risk factor for FVIII inhibitor development in severe HA patients. We studied 44 patients from 31 severe HA families for the detection of inv22 and 94 patients from 65 families to detect inv1. We used the subcycling long-distance PCR to detect inv22 and rapid PCR in duplex reactions to detect inv1. We found a frequency of 45% for the inv22 and no inv1-positive patients (0%). These frequencies were not statistically different from other populations, although haplotype analyses of FVIII gene and telomeric regions should be incorporated to explore population-specific variation of inv1 frequencies. Inv22-positive patients showed 1.88X higher risk for developing inhibitors with respect to patients carrying other severe mutations; however, this OR value was not significant. Our findings confirm inv22 as a hot-spot for severe HA and evidence the low frequency of inv1 in a Mexican population. The non-significant risk for developing inhibitors among inv22-positive patients agrees with the variety of genetic and non-genetic factors involved in such a complication. Am. J. Hematol. 82:283-287, 2007. V V C 2007 Wiley-Liss, Inc.

In Mexico, 15% of haemophilia A (HA) patients develop inhibitory alloantibodies in response to replacement therapy with factor VIII (FVIII), requiring bypass therapy such as activated prothrombin complex concentrate (APCC). Because bypass therapy has not been broadly available in Mexico even in recent years, this study aimed to evaluate the thrombin generation assay (TGA) in assessing the response to FVIII or APCC treatment in patients with severe HA positive to inhibitors. We studied 189 patients with severe HA. Clinical severity was verified by one-stage APTT-based clotting assay. Inhibitors to FVIII were investigated by the Nijmegen-Bethesda (N-B) method, and type of inhibition was assessed through serial plasma dilutions. Thrombin generation was measured with the calibrated automated thrombogram in inhibitor-positive plasmas previously spiked and incubated with FVIII or APCC. Data were analysed using anova, Student or Fisher's exact tests. We detected 47 (24.9%) subjects with high-titre (5-1700 N-B U mL(-1)) and 25 (13.2%) subjects with low-titre inhibitor antibodies (0.6-4.7 N-B U mL(-1)). We found an association between kinetic behaviour and clinical response to FVIII (P = 0.0049) or vs. FVIII response evaluated with TGA (P = 0.0007). Global concordance between clinical and in vitro response was 70%. By evaluating the capacity of thrombin formation in a plasma sample, TGA predicts the response to FVIII or APCC therapy and allows individual optimization of resources in patients with severe HA and high-titre inhibitors. The inhibition pattern of the antibodies to FVIII:C correlated with the TGA parameters and showed an association with the clinical response to FVIII.