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Introduction: Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease., Materials and Methods: We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined., Results: Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function., Conclusions: Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

It is widely accepted that blood coagulation in vivo is initiated during normal hemostasis, as well as during intravascular thrombus formation, when the cell-surface protein, tissue factor (TF), is exposed to the blood as a consequence of vascular injury. In addition to its essential role in hemostasis, tissue factor may be also implicated in several pathophysiological processes, such as intracellular signaling, cell proliferation, and inflammation. For these reasons, the tissue factor:factor VIIa complex has been the subject of intense research focus. Many experimental studies have demonstrated that inhibition of tissue factor:factor VIIa procoagulant activity are powerful inhibitors of in vivo thrombosis and that this approach usually results in less pronounced bleeding tendency, as compared to other "more classical" antithrombotic interventions. Alternative approaches may be represented by transfecting the arterial wall with natural inhibitors of tissue factor:factor VIIa complex, such as tissue factor pathway inhibitor (TFPI), which may result in complete inhibition of local thrombosis without incurring in potentially harmful systemic effects. Additional studies are warranted to determine the efficacy and safety of such approaches in patients., (Copyright 2002 Elsevier Science Ltd.)

We have found that synthetic peptides derived from the two epidermal growth factor-like domains of factor VII are inhibitors of tissue factor dependent factor X activation. Inhibition was most pronounced for a constrained sequence of amino acids corresponding to positions 91-102 of factor VII, Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gin-Tyr-Cys. The biological activity appeared to be localized to the tripeptide 'motif', Glu-Gln-Tyr, within the larger sequence. The cyclic peptide was also an inhibitor of tissue factor induced coagulation of plasma, using lipidated tissue factor or tissue factor expressed on the surface of living cells. However, it did not interfere with intrinsic coagulation. Inhibition of factor X activation was dose-dependent with an IC50 value of 350 microM. Kinetic analyses revealed non-competitive inhibition with respect to factor X and suggested that the peptide sequence interferes with the factor VII/tissue factor/factor X complex formation and function. A pentapeptide analog of the putative pharmacophore was also a dose-dependent inhibitor of factor X activation with an IC50 value of 560 microM, but the tripeptide, Glu-Gin-Tyr, alone was without effect. Our results suggest a direct role for the second epidermal growth factor-like domain of factor VII, and in particular its loop I, in the formation and function of the factor VII/tissue factor/factor X complex.

Recent studies have suggested that retarded fetal and early growth predisposes to cardiovascular disease of males in adult life. This is partly explicable by an association between the haemostatic variables fibrinogen and factor VII and the placenta/birth weight ratio. Since many characteristics influence clotting factors during life, levels of fibrinogen and clotting factor VII were measured in cord blood of male neonates. The placenta/birth weight ratio did not correlate with the birth weight for gestational age and appeared to be only a minor indicator of fetal growth. These findings make it unlikely that the level of fetal outcome 'programmes' male neonates via fibrinogen or factor VII to develop cardiovascular disease in adult life.

Colburn and Buonassisi (In Vitro Cell Dev. Biol. 24, 1133-1136, 1988) have isolated a single chain sulfated glycoprotein inhibitor of factor VIIa/tissue factor-catalyzed activation of factor X from conditioned media of an established rabbit endothelial cell line. We report herein that their endothelial cell-derived inhibitor and extrinsic pathway inhibitor (EPI) isolated from rabbit plasma have identical functional properties with respect to their interactions with factor Xa and with factor VIIa/tissue factor. In addition, the endothelial cell inhibitor and rabbit plasma EPI migrate with the same apparent molecular weights on non-reduced SDS-PAGE and contain similar amounts of N-linked carbohydrate. Like the endothelial cell inhibitor the EPI of rabbit plasma exists as a single chain molecule. Furthermore, the endothelial cell inhibitor is recognized and neutralized by a polyclonal antibody raised against rabbit plasma EPI. We therefore conclude that cultured rabbit endothelial cells produce an inhibitor of factor VIIa/tissue factor activity that is functionally and immunologically identical to rabbit plasma EPI.

Tissue factor activity expressed by Hela cells cultured in 96-well plates has been quantitated in situ using a continuous spectrophotometric assay. Following the assay, cells assayed without physical disruption remained as viable as cells not subjected to the assay. Very little (or no) tissue factor was expressed in nondisrupted cells relative to that available in cells disrupted by freeze-thawing and sonication. Total tissue factor activity (that available in disrupted cells) decreased not as a simple function of time after subculturing, but was inversely related to cell density.

Partially purified Amniotic Fluid Factor (AFF) is found to directly activate human factor X, with a Km for factor X of 0.37 +/- 0.06 M. Added phospholipid has only a slight effect on the activation at low concentrations, and inhibits the reaction at higher concentrations. Both DIPF and PMSF inhibit AFF factor X activation. Although added phospholipid is not required for AFF-activity, phospholipase C rapidly destroys it, indicating the presence of intrinsic phospholipid. Phospholipase C treated AFF releases factor VII activity, which leads to the conclusion that AFF is in fact a thromboplastin: factor VII complex. Both AFF and a human brain thromboplastin factor VII complex prepared in vitro were inhibited by Zn++ ion, while human brain thromboplastin alone is not. AFF is markedly larger than the human brain thromboplastin-factor VII complex as judged by gel filtration.

Factors affecting the inhibition of tissue thromboplastin (TP)-mediated blood coagulation have been investigated. Human brain thromboplastin progressively loses procoagulant activity when incubated in the presence of defibrinated plasma and CaCl2. Inhibition is maximal at a CaCl2 concentration of 1.5 mM during incubation and involves the calcium dependent binding of a plasma component(s) to the TP-FVII complex, preventing the activation of FX. Chelation of calcium ions using EDTA releases active TP and FVII from the inhibited complex. No inhibition occurs during incubation of TP with Al (OH)3 adsorbed plasma and calcium ions unless a Factor VII concentrate (or purified FVII and FX) is also present. Incubation of TP with antithrombin III-deficient plasma and calcium ions also leads to inhibition. Moreover, purified AT III cannot substitute for adsorbed plasma in producing TP inhibition. The data are consistent with the presence in plasma of a potent AT III independent inhibitor of TP-mediated blood coagulation.