Biomedical subjects

James H Morrissey

Publications and source records attributed to James H Morrissey.

4 recordsLinked to original sources

PubMed2004

The cofactor function of the N-terminal domain of tissue factor.

Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF(2-219) in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N- and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg(74) mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.

Allosteric Regulation↗

PubMed2004

Tissue factor: a key molecule in hemostatic and nonhemostatic systems.

Tissue factor (also known as tissue thromboplastin or CD142) is the protein that activates the blood clotting system by binding to, and activating, the plasma serine protease, factor VIIa, following vascular injury. Because of its essential role in hemostasis, tissue factor plays a role in pathology associated with hemostasis, triggering the coagulation system in many thrombotic diseases and the coagulopathies associated with sepsis and other forms of disseminated intravascular coagulation. Recent research has also implicated tissue factor in a variety of nonhemostatic roles, including cell signaling, inflammation, vasculogenesis, and tumor growth and metastasis. This review focuses on both the well-known roles of tissue factor in hemostasis and thrombosis and the newer concepts of tissue-factor biology including how it functions as a signaling receptor and the possible role of blood-borne tissue factor in thrombosis.

Animals↗

PubMed2002

Tissue factor alters the pK(a) values of catalytically important factor VIIa residues.

Blood coagulation is triggered when the serine protease factor VIIa (fVIIa) binds to cell surface tissue factor (TF) to form the active enzyme-cofactor complex. TF binding to fVIIa allosterically augments the enzymatic activity of fVIIa toward macromolecular substrates and small peptidyl substrates. The mechanism of this enhancement remains unclear. Our previous studies have indicated that soluble TF (sTF; residues 1-219) alters the pH dependence of fVIIa amidolytic activity (Neuenschwander et al. (1993) Thromb. Haemostasis 70, 970), indicating an effect of TF on critical ionizations within the fVIIa active center. The pKa values and identities of these ionizable groups are unknown. To gain additional insight into this effect, we have performed a detailed study of the pH dependence of fVIIa amidolytic activity. Kinetic constants of Chromozym t-PA (MeSO(2)-D-Phe-Gly-Arg-pNA) hydrolysis at various pH values were determined for fVIIa alone and in complex with sTF. The pH dependence of both enzymes was adequately represented using a diprotic model. For fVIIa alone, two ionizations were observed in the free enzyme (pK(E1) = 7.46 and pK(E2) = 8.67), with at least a single ionization apparent in the Michaelis complex (pK(ES1) similar 7.62). For the fVIIa-TF complex, the pK(a) of one of the two important ionizations in the free enzyme was shifted to a more basic value (pK(E1) = 7.57 and pK(E2) = 9.27), and the ionization in the Michaelis complex was possibly shifted to a more acidic pH (pK(ES1) = 6.93). When these results are compared to those obtained for other well-studied serine proteases, K(E1) and K(ES1) are presumed to represent the ionization of the overall catalytic triad in the absence and presence of substrate, respectively, while K(E2) is presumed to represent ionization of the alpha-amino group of Ile(153). Taken together, these results would suggest that sTF binding to fVIIa alters the chemical environment of the fVIIa active site by protecting Ile(153) from deprotonation in the free enzyme while deprotecting the catalytic triad as a whole when in the Michaelis complex.

Catalysis↗

PubMed2002

Tissue factor as a proinflammatory agent.

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4-Mac1+ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1alpha and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.

Animals↗