Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex.

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But approximately 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.

A novel mutation (deletion of Aalpha-Asn 80) in an abnormal fibrinogen: fibrinogen Caracas VI. Consequences of disruption of the coiled coil for the polymerization of fibrin: peculiar clot structure and diminished stiffness of the clot.

An abnormal fibrinogen was identified in a 10-year-old male with a mild bleeding tendency; several years later, the patient developed a thrombotic event. Fibrin polymerization of plasma from the propositus and his mother, as measured by turbidity, was impaired. Plasmin digestion of fibrinogen and thrombin bound to the clot were both normal. The structure of clots from both plasma and purified fibrinogen was characterized by permeability, scanning electron microscopy and rheological measurements. Permeability of patients' clots was abnormal, although some measurements were not reliable because the clots were not mechanically stable. Consistent with these results, the stiffness of patients' clots was decreased approximately two-fold. Electron microscopy revealed that the patients' clots were very heterogeneous in structure. DNA sequencing of the propositus and his mother revealed a new unique point mutation that gives rise to a fibrinogen molecule with a missing amino acid residue at Aalpha-Asn 80. This new mutation, which would disrupt the alpha-helical coiled-coil structure, emphasizes the importance of this part of the molecule for fibrin polymerization and clot structure. This abnormal fibrinogen has been named fibrinogen Caracas VI.

Radiosynoviorthesis in haemophilia: how safe?

One of the best procedures to prevent haemarthrosis in haemophilia has been radioactive synovectomy (radiosynoviorthesis). Since 1976 we have performed 119 radiosynoviortheses in 110 patients, aged from 3 to 40 years (mean 10), and of whom 71 were under 12 years of age. The knees were injected in 71, elbow in 29, ankles in 16, and shoulders in 3 cases. Clinical results of the procedure gave excellent results 80% of patients with no further bleeding. In the case of failure a reinjection can be given in the same joint at a 6 month interval. One of the criticisms against this method is possible chromosomal damage. In our centre, 4 studies have been made in order to see whether these changes are permanent, but all have demonstrated that chromosomal changes are reversible. Radioactive material used in 2 studies was Au-189. In 1978, 354 metaphases were studied with 61 ruptures, with 17.23% non-premalignant and 6 structural changes considered premalignant (1.69%). Further study was done in 1982, in the same group of patients with the result of 21 ruptures (3.34%) and no structural changes. The third study was performed in 13 patients that sustained radiosynoviorthesis with Re-186 in 1991. We compared the chromosomal study before and 6 months after the radioactive material injection and the results confirmed that changes appeared equally in non-irradiated and radiated patients and disappeared with time, never reaching the dangerous zone of 2%. In the group treated with Re-186 we studied an additional number of 130 metaphases with identical results and no structural changes. A study performed before and after radiosynoviorthesis with Y-90 revealed no premalignant changes. It seems than radiosynoviorthesis is safe and highly beneficial to haemophilic patients.

Factor V Leiden and the risk of venous thrombosis, myocardial infarction, and stroke: a case-control study in Venezuela.

The most common genetic defect associated with deep vein thrombosis (DVT) is a mutation in the Factor V gene (G1691A), known as Factor V Leiden (FVL). We investigated the genotypes for FVL in 571 individuals in Venezuela: 208 patients with DVT, 175 patients with acute myocardial infarction, 54 patients with stroke, and 134 control subjects. Our results showed in the population analyzed here that the FVL was associated with a fourfold increase in the risk for DVT (odds ratio, 4.24; 95% confidence interval, 1.35-14.79); particularly, women carriers showed a 6.5-fold increase in the risk for DVT. No relation was observed between the presence of FVL and the risk for acute myocardial infarction or stroke. In conclusion, a clear association between the FVL mutation and DVT was observed in the population analyzed in Venezuela. These results are in agreement with those found in other populations with different ethnic backgrounds.

Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.