The receptor for urokinase plasminogen activator (uPA) has been previously shown not to internalize its ligand, but rather to focalize its activity at the cell surface, allowing a regulated cell surface plasmin dependent proteolysis. The receptor in fact binds the proenzyme pro‐uPA and allows its very efficient conversion to the active two chains form. Receptor bound active uPA can also interact with its specific type 1 inhibiror (PAI‐1) which is therefore able to inhibit the cell surface plasmin formation. In this paper we show that the uPA‐PAI‐1 complex bound to the uPA receptor is internalized and degraded. U937 cells were incubated at 4 degrees C with labeled uPA‐PAI‐1 (and other ligands), the temperature then raised to 37 degrees C and the fate of the ligand followed for 3 h thereafter. The uPA‐PAI‐1 complex was internalized into the cells (i.e. could not be dissociated by acid treatment) and thereafter degraded (i.e. appeared in the supernatant in a non TCA‐precipitable form). Other ligands (free uPA, ATF and DFP‐treated uPA) were not internalized nor degraded. The degradation of the uPA‐PAI‐1 complex is preceded by internalization and is inhibited by chloroquine, an inhibitor of lysosomal protein degradation. These data suggest the existence of a cellular cycle of uPA. After synthesis pro‐uPA is secreted, bound to the receptor and activated to two chain uPA. On the surface, uPA can activate surface bound plasminogen to produce surface bound plasmin. In the presence of PAI‐1 uPA activity is inhibited and plasmin production interrupted, while the uPA‐PAI‐1 complex is internalized and degraded.

The inhibition by tissue factor pathway inhibitor (TFPI) of its two target enzymes--factor Xa and the tissue factor-factor VIIa complex (TF:VIIa)--has been studied under near-physiological reactant concentrations and conditions. Over a TFPI range of 0-1 nM, the rate of inhibition of factor Xa, in the presence of Ca2+ and anionic phospholipid vesicles at 37 degrees C, was proportional to TFPI concentration, giving an association rate, k1, of 0.96 x 10(9) M-1 min-1. Factor Xa inhibition did not proceed to completion, the reaction attaining a near-equilibrium that was dependent on the TFPI concentration. The estimated dissociation rate of the TFPI:Xa complex, k-1, was independent of TFPI concentration, with a mean value of 0.02 min-1. The resulting calculated value of K1, the apparent dissociation constant for the initial step, is 21 pM. Slow decay of the remaining factor Xa in such incubations, detectable after attainment of the rapid initial near-equilibrium, confirmed the two-step mechanism proposed by Huang et al. (1993) [J. Biol. Chem. 268, 26950-26955], but did not permit determination of a rate constant for the second step. Omission of anionic phospholipid had no significant effect on either k1 or k-1. A high-molecular-weight fraction of heparin, at saturating levels (> or = 0.05 unit/mL, congruent to 25 nM), increased k1 2-fold, with no detectable effect on k-1. The second stage of TFPI action was studied by preformation of the TFPI:Xa complex, and its incubation with the TF:VIIa complex in the presence of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

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To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.

BACKGROUND This study was designed to determine whether arterial reocclusion after thrombolysis can be prevented by lipoprotein-associated coagulation inhibitor (LACI), a physiological inhibitor of tissue factor-induced coagulation mediated by the extrinsic pathway. METHODS AND RESULTS Thrombosis was induced in femoral arteries of anesthetized dogs with the use of anodal current to elicit extensive vascular injury and formation of platelet-rich thrombi in one artery and with thrombogenic copper wire to elicit fibrin-rich thrombi without appreciable vascular injury in the contralateral artery. Recanalization of both vessels was induced with t-PA (1.7 mg/kg i.v. over 1 hour) and verified with Doppler flow probes. Reocclusion occurred within 2 hours in seven of seven arteries with electrical injury-induced thrombosis and in four of seven arteries with copper wire-induced thrombosis in the absence of LACI. In dogs given infusions of recombinant DNA-produced LACI (225 micrograms/kg over 15 minutes, followed by 4 micrograms/kg/min i.v.) after completion of the infusion of t-PA, no reocclusion occurred during the 2-hour interval of observation in any of the five arteries subjected to electrical injury (p less than 0.001), and cyclic partial occlusions were nearly abolished (0.4 +/- 0.4/hr in LACI-treated dogs compared with 13.7 +/- 5.5/hr in saline-treated dogs, p less than 0.0001). In contrast, reocclusion occurred in two of five arteries with indwelling copper wires, and cyclic partial occlusions were unaffected despite LACI. LACI prolonged the partial thromboplastin time modestly (1.7 +/- 0.2 x baseline) but did not affect platelet counts or aggregation assessed ex vivo. CONCLUSIONS Inhibition of the extrinsic pathway of coagulation with LACI prevents thrombotic arterial reocclusion after thrombolysis in vessels subjected to extensive vascular injury. Our results demonstrate that activation of the extrinsic pathway plays a critical role in thrombotic reocclusion and that LACI provides a highly targeted approach to facilitate sustained recanalization without directly inhibiting platelets.