Abstract
Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor. It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation. Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction. The inactivation of factor Xa by E. coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process. The slow, tight-binding inhibition of factor Xa follows the scheme: [formula: see text] Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex. In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E. coli-rTFPI are 1.24 nM and 26.4 pM, respectively. In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM. The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki. In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM. The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III. At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.
Highlights
From the j9ivision of HematologylOncology, Jewish Hospital at Washington University Medical Centel; St
The enzymds) responsible for this proteolysis has not been identified, the cleavage occurs within a stretch of basic amino acid residues (TFPI residues 254-261) and produces a truncated form of TFPI that lacks the carboxyl-terminal 15-20 amino acids of full-length TFPI and thatpossesses considerably less factor Xa inhibitory activity [8].Our laboratory has previously reported a preliminary analysis of the kinetics of factor Xa inhibition by TFPI produced in the humanhepatoma cell line HepG2 [5]
Subsequentstudies, revealed that the HepG2-TFPI used for those studies was truncated at the carboxyl terminus.sufficient full-length rTFPIwas produced in a prokaryotic expression system to repeat thekinetic analysis of factor Xa inhibition
Summary
From the j9ivision of HematologylOncology, Jewish Hospital at Washington University Medical Centel; St. In the presence of calcium ions proteolyticcleavage of TFPI produced by human leukocyte elastase that separates the amino terminus and first Kunitztype domain from the second Kunitz-type domain and the remainder of the molecule [9].carboxyl-terminal trun-. (5 m ~ th)e interaction between factor Xa and rTFPI is cation of the TFPI molecule markedly reduces its factor Xa substantially weaker, witha Ki of 42.7 n~ and Ki*of 85.2 inhibitory activity [8].Such carboxyl-terminal truncation has PM.The addition of other components of the prothrom- been demonstrated in recombinant TFPIproduced by cells in binase complex producesenhanced factor Xa inhibition tissue culture [8, 10]. We report the kinetics of factor Xa inhibition by full-length rTFPI in the presence and absence of components of the prothrombinasecomplex and heparin
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