Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in hemophilia A, hemophilia B and factor VII-deficient plasmas.

Carbon monoxide derived from carbon monoxide releasing molecules (CORMs) has been demonstrated to enhance normal plasma thrombus speed of growth and strength in vitro. We tested the hypothesis that tricarbonyldichlororuthenium (II) dimer (CORM-2) improves the velocity of formation and strength of hemophiliac plasma thrombi as determined by thrombelastography. Plasma deficient (<1% normal activity) in factor VIII (FVIII; n = 11 individuals), factor IX (FIX; n = 5 individuals) or factor VII (FVII; n = 4 individuals) was exposed to 0 or 100 micromol CORM-2, with coagulation initiated with tissue factor. Coagulation kinetics were monitored with thrombelastography for 15 min. Paired t-tests were used to analyze FVIII-deficient plasma results; relative change was used to describe the other plasma types tested. In FVIII-deficient plasma, CORM-2 exposure significantly (P < 0.05) increased the velocity of thrombus formation (84%) and strength (48%) compared with plasma not exposed to CORM-2. FXI-deficient clots demonstrated an increase in velocity of formation (63%) and strength (43%) after CORM-2 exposure. Lastly, CORM-2 exposure increased FVII-deficient plasma velocity of formation (45%) and strength (63%). CORM-2 markedly enhanced the velocity of clot growth and strength in hemophiliac plasma. These findings serve as the rationale to determine whether CORMs could be utilized as hemostatic agents.

Severe factor VII deficiency caused by a novel mutation His348 to Gln in the catalytic domain.

Factor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient's factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule.

Phospholipase C-sensitive factor VII complexes in dog plasma.

We report the presence of a phospholipase c-sensitive activated factor VII complex in canine plasma after feeding a special diet. Low levels of complex was observed in the fasting state. The response to feeding in terms of activated factor VII varied markedly among the dogs investigated.

Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5-8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded.