Calpains are a family of Ca 2؉ -dependent intracellular cysteine proteases, including the ubiquitously expressed -and m-calpains. Both -and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of -and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both -and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.

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To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312؊19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312؊19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD. (Blood. 2003;102:549-557)

Summary. Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family-based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ÔOÕ and ÔAÕ alleles and the VWF codon 1584 variant. There was also weak association with the )1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

ABSTRACT

The bacterial production of recombinant rat calpain II has been improved greatly by the use of two compatible plasmids for the two subunits. The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements into pACYC177. This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these two vectors. A high level of activity was also obtained from a bicistronic construct containing both subunit cDNAs under the control of one T7 promoter. The addition of a C-terminal His-tag to the large subunit simplified purification without affecting subunit association or enzyme activity. The active-site cysteine 105 was mutated to alanine, causing complete loss of activity. The yield of purified C105A-calpain II (80 + 21 kDa) dimer following three column chromatography steps was 10 mg/l of cell culture. This provides a purified calpain, stable to autolysis and oxidation, which is likely to facilitate crystallization in both the presence and absence of calcium.

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca 2؉ The calpains (EC 3.4.22.17) are cytoplasmic cysteine proteinases, which are thought to be regulated by means of their Ca 2ϩ dependence. While much work has been done on their biochemical properties, many aspects of autolysis, activation, and Ca 2ϩ requirement remained unresolved (1-3). Two mammalian forms, -and m-calpain (calpain I and II), have been most studied, since they can be isolated from animal tissues. Some other calpain forms are known so far only from their mRNA (3, 4), and the chicken calpains are not further considered here since they appear to be slightly different (5, 6). Complete purification of calpain in adequate amounts from tissue extracts is difficult, and a bacterial expression system has been described, which with the aid of a His-tag provides larger amounts of pure enzyme in about 3 days and provides a means for mutational and structural work (7). A baculovirus-based expression system has also been reported (8).The calpains consist of an 80-kDa catalytic subunit (the large subunit), containing four domains, I-IV, and a 28-kDa regulatory subunit (the small subunit), containing two domains, V and VI (9, 10). Domains I and V are involved in autolysis; domain II contains the most obvious active site residues (11); the function of domain III is not yet known, although it must also take part in the conformational changes induced by Ca 2ϩ ; and the C-terminal domains of both subunits, IV and VI, contain putative E-F hand motifs, some of which bind Ca 2ϩ (12)(13)(14)(15). No structural information is available for the complete calpain heterodimer, but the crystal structures of domain VI, with and without bound Ca On exposure to sufficient Ca 2ϩ , the calpains are assumed to undergo a conformational change which permits the following four events: limited autolysis of the small subunit from 28 to 20 kDa; limited N-terminal autolysis of the large subunit; proteolysis of a substrate such as casein if it is present; and further inactivating proteolysis of the large subunit. However, the order of these events and the Ca 2ϩ concentrations required have been difficult to establish. The Ca 2ϩ requirements ([Ca 2ϩ ] 0.5 ) for casein hydrolysis by (initially) non-autolyzed calpains are usually reported to lie in the ranges of 5-50 M Ca 2ϩ for -calpain and 250 -1000 M Ca 2ϩ for m-calpain. These ranges are lowered by prior autolysis to 1-5 M Ca 2ϩ for -calpain and 100 -200 M Ca 2ϩ for m-calpain (16 -25). The reported values vary widely for at least two reasons, first because the casein assay reflects the net effects of activation, autolysis, and inactivation, and is not well suited for kinetic studies (26); and second because, as shown here, the observed values are highly dependent on experimental conditions. While it is fairly clear that autolysis lowers [Ca 2ϩ ] 0.5 of both -and m-calpain, it has not been clear whether large and/or small subunit cleavage is req...

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.