Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity ofFactor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserinespecific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex. (J. Clin. Invest. 1992. 90:2340-2347

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Development of distinct CD4+ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double-label in situ hybridization technique was used to characterize co-expression of cytokine mRNA in antigen-specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4+ T cells isolated from alpha beta T cell receptor-transgenic mice. Interleukin (IL)-2 was the dominant cytokine expressed by antigen-stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)-gamma were less frequent, and IL-4, IL-5, and IL-10 were infrequent. In contrast, cells that expressed IFN-gamma or IL-10 were most frequent in the LP population, with lower frequencies of IL-2, and few IL-4- and IL-5-positive cells. Co-expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene-positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4+ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4+ T population has a distinct cytokine expression pattern with counter-regulatory properties that may be important for homeostasis in mucosal immune tissues.

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The amino terminal sequence of the Candida albicans cell wall protein Int1 exhibited partial identity with the major histocompatibility complex (MHC) class II binding site of the Mycoplasma arthritidis superantigen MAM. Int1-positive C. albicans blastospores activated human T lymphocytes and expanded Vbeta subsets 2, 3, and/or 14; Int1-negative strains were inactive. Release of interferon-gamma (IFN-gamma) but not of tumor necrosis factor-alpha or interleukin-6 was Int1 dependent; interleukin-4 and interleukin-10 were not detected. T lymphocyte activation, Vbeta expansion, and IFN-gamma release were associated with a soluble polypeptide that encompassed the first 263 amino acids of Int1 (Pep(263)). Monoclonal antibody 163.5, which recognizes an Int1 epitope that overlaps the region of identity with MAM, significantly inhibited these activities when triggered by Int1-positive blastospores or Pep(263) but not by staphylococcal enterotoxin B. Histidine(263) was required. Pep(263) bound to T lymphocytes and MHC class II and was detected in the urine of a patient with C. albicans fungemia. These studies identify a candidal protein that displays superantigen-like activities.

Although there are significant differences in the frequency of different cellular subsets and patterns of cytokine gene expression, these differences are quantitatively subtle, suggesting a delicately balanced immune response that can develop a pattern of specific unresponsiveness, with relatively minor alterations in the specific T-cell response.

A simple 30-min enzyme digestion procedure has been used to release guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture. Primary cell cultures initially consist of numerous epithelial colonies and 70-1000 morphologically differentiated smooth muscle cells per 600 mg (wet weight) tracheal tissue depending on the age of the animal. Both cell types proliferate to form a confluent monolayer within 5-17 days. Pure subcultures of tracheal smooth muscle cells are obtained by limited trypsin digestion of the primary culture. Eighty percent of these subcultured smooth muscle cells retain the ability to contract in response to histamine (10(-6) M) and to form reaggregates even after 20 or more passages. Examination of these cells by electron microscopy reveals both biosynthetic and contractile components of smooth muscle. Analysis of this dual phenotype may provide valuable information about the regulation of tracheal smooth muscle cell growth and differentiation.