Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.

Objective-The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). T ightly controlled exposure of tissue factor (TF) to components of the plasma coagulation cascade is important for maintenance of normal rheological properties of blood. Failure to manipulate TF levels available for the initiation of blood clotting leads to thrombotic or bleeding disorders in humans. Circulating monocytes are presumably the major cell type that respond to variable stimuli by developing coagulant activity 1 through the expression of TF. 2 Originally, intersubject variability in developing of monocyte TF activity was described by Østerud et al. 3 By comparing lipopolysaccharide (LPS)-induced monocyte TF activity and tumor necrosis factor-␣ (TNF-␣) production in a whole blood system, an up to a 50-fold difference between individuals was observed. 4 This finding was defined as the "highlow responder phenomenon." 4 Also noteworthy, the individual usually remains a high responder (HR) or low responder (LR) for several years. 5,6 Later, high intersubject variability in cytokine production by LPS-stimulated monocytes was demonstrated. 7 It was also shown that patients with high levels of TNF-␣ production were more susceptible to heart transplant rejection. 8 Monocytes isolated from septic shock patient survivors revealed higher TNF-␣ production than monocytes from nonsurvivors. 9 Many studies have been undertaken to describe the significance of this phenomenon, but so far, no general explanation has been found. Diverse plasma factors and direct cell interactions play an important role in the development of monocyte TF activity. 10 -15 High expression of monocyte TF activity is associated with higher risk of acute coronary syndrome. 16 Platelets have been suggested to be responsible for inducing monocyte TF activity. 17 Platelet-rich plasma induced significantly higher TF activity in LPS-stimulated monocytes than platelet-poor plasma. 18 Moreover, when blood cells without platelets from HRs were mixed with platelet-rich plasma of an LR, LPS-induced TF activity was reduced up to 76% compared with an autologous system. 18 It was shown that granulocytes enhance LPSinduced monocyte TF activity in a platelet-dependent reaction involving P-selectin, platelet factor 4, plateletactivating factor, hydroxyl-eicosatetraenoic acid, and platelet-derived growth factor. 18 -21 Here we report several observations concerning the relationships between intracellular-and membrane-located TF antigen in resting and LPS-stimulated monocytes in groups of HRs and LRs, using fluorescence-activated cell sorter (FACS) analysis, fluorescence confocal microscopy, in-cell Methods and Results-After Materials and Methods Blood Sampling and Experimental DesignBlood samples from 16 healthy ...

Summary. The large number of conflicting reports on the presence and concentration of circulating tissue factor (TF) in blood generates uncertainties regarding its relevance to hemostasis and association with specific diseases. We believe that the source of these controversies lies in part in the assays used for TF quantitation. We have developed a highly sensitive and specific double monoclonal antibody fluorescence-based immunoassay and integrated it into the Luminex Multi-Analyte Platform. This assay, which uses physiologically relevant standard and appropriate specificity controls, measures TF antigen in recombinant products and natural sources including placenta, plasma, cell lysates and cell membranes. Comparisons of reactivity patterns of various full-length and truncated TFs on an equimolar basis revealed quantitative differences in the immune recognition of TFs by our antibodies in the order of TF 1-263 > 1-242 > 1-218 > placental TF. Despite this differential recognition, all TF species are quantifiable at concentrations 6 2 pM. Using a calibration curve constructed with recombinant TF 1-263 and plasma from healthy individuals (n ¼ 91), we observed the concentration of TF antigen in plasma to be substantially lower than that generally reported in the literature: TF antigen in plasma of 72 individuals (79%) was below 2 pM (quantitative limit of our assay); TF antigen levels between 2.0 and 5.0 pM could be detected in six individuals (7%); and in 14% (13 plasmas), the non-specific signal was higher than the specific signal, and thus TF levels could not be determined. These differential recognition patterns affect TF quantitation in plasma and should be considered when evaluating plasma TFlike antigen concentrations.

Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH 2 -terminal ''catalytic switch'' that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSK⌬59) fusion protein lacking the 59 NH 2 -terminal residues was no longer capable of fibrinindependent Pg activation (k cat ͞K m decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH 2 -terminal peptide rSK1-59. Deletion of the NH 2 terminus made rSK⌬59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSK⌬59 activator complex apparently resulted from selective catalytic processing of fibrinbound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSK⌬59) of the SK NH 2 -terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSK⌬59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH 2 terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH 2 terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.