Although the majority of factor VII (FVII) circulates in the zymogen form, low levels of activated factor VII (FVIIa) have been postulated to exist in plasma and to serve a priming function for triggering of the clotting cascade. However, direct measurement of plasma FVIIa has not previously been possible. We have quantified plasma FVIIa levels using a novel, highly sensitive assay that is free from interference by FVII. Specificity of this clot-based assay results from the use of a mutant tissue factor that is selectively deficient in promoting FVII activation, but retains FVIIa cofactor function. In normal adults, FVIIa was found to be present in plasma (mean: 3.6 ng/mL) with considerable variation between individuals (range: 0.5 to 8.4 ng/mL). FVIIa levels were only loosely correlated with FVII coagulant activity, but were elevated in pregnancy and reduced with oral anticoagulant therapy. Incubation of plasma on ice in glass containers (cold activation) resulted in substantial FVIIa generation. Measurement of plasma forms of factor VII is of potential clinical importance because elevated FVII coagulant activity has been implicated as a significant risk predictor for ischemic heart disease. Clinically, this new assay will now permit direct assessment of the role of plasma FVIIa in thrombotic disorders.

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8–11D12 and TF9–9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9–9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8–11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8–11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8–11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

In order to examine the control of human factor X biosynthesis we have molecularly cloned the cDNA and investigated the expression of the Factor X gene. A recombinant clone of approximately 1100 base pairs in length containing the sequence of factor X was identified in a lambda gt11 human liver cDNA library by screening with polyclonal antibodies. One plaque was selected and confirmed for specificity with a mixture of five factor X specific monoclonal antibodies (MoAbs). A partial nucleic acid sequence of the 5′ end of the cDNA corresponded to the described amino acid sequence between residues 41 and 56 of the light chain of factor X. Northern blot analysis of RNA from human liver and the hepatoma cell line, Hep G2, identified the factor X mRNA as a single molecular species of approximately 1700 bases. Cell lines which do not secrete factor X did not contain factor X mRNA indicating restriction of transcription to hepatocytes. Slot-blot hybridization analysis of factor X and actin mRNA demonstrated no change in the levels of total or specific factor X mRNA in Hep G2 cells following treatment with warfarin or vitamin K. We conclude that modulation of factor X production by these drugs, known to influence gamma-carboxylation and total factor X secretion by these cells, is mediated by changes in posttranscriptional events rather than by effects on the steady state levels of factor X mRNA.

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