The binding of low molecular weight heparin to hemostatic enzymes.

Jordan, Robert E.; Oosta, G M; Gardner, W.T.; Rosenberg, R D

doi:10.1016/s0021-9258(19)70430-x

Cited by 102 publications

(15 citation statements)

References 29 publications

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“…rTFPI-C as shown in Figure IB. The value is comparable with those for the interaction of heparin with thrombin (8.0 x Hr7 M)(Jordan et al, 1980), factor IXa (2.6 x 1()"7 M)(Jordan et al, 1980), factor Xa (8.7 x 10~6 M)(Jordan et …”

supporting

confidence: 76%

Effect of Heparin on the Inhibition of Factor Xa by Tissue Factor Pathway Inhibitor: A Segment, Gly212-Phe243, of the Third Kunitz Domain Is a Heparin-Binding Site

Enjyoji¹,

Miyata²,

Kamikubo³

et al. 1995

Biochemistry

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Tissue factor pathway inhibitor (TFPI) inhibits the tissue factor--factor VIIa complex and factor Xa with its first and second Kunitz domains (K1 and K2), respectively. The inhibitory activity is enhanced by heparin, and the C-terminal basic part has been shown to be a heparin-binding site (HBS-1). To characterize and localize a second heparin-binding site (HBS-2), we studied the effect of heparin on the inhibitory activity of two forms of recombinant human TFPI, the full-length TFPI (rTFPI), and TFPI lacking the C-terminal basic part (rTFPI-C), by assaying the inhibition of human factor Xa. rTFPI-C inhibited factor Xa with an initial Ki of 6.79 nM in the absence of Ca2+ and 22.3 nM in the presence of 5 mM CaCl2. Heparin decreased the initial Ki to 1.79 nM in the absence of Ca2+ and 2.68 nM in the presence of 5 mM CaCl2, indicating the presence of HBS-2 in rTFPI-C. The dissociation constant for the binding of HBS-2 with heparin was determined to be 830 nM using fluorescein-labeled heparin and rTFPI-C. Heparin enhanced the inhibitory activity of a fragment consisting of the K2 and K3 domains, but it did not stimulate the inhibitory activity of the K2 domain. A synthetic peptide mimicking from Gly212 to Phe243 in the K3 domain reduced the effect of heparin on the inhibition by rTFPI-C and rTFPI. These results defined the location of HBS-2 in the basic region of the K3 domain between Gly212 and Phe243.

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supporting

confidence: 76%

Effect of Heparin on the Inhibition of Factor Xa by Tissue Factor Pathway Inhibitor: A Segment, Gly212-Phe243, of the Third Kunitz Domain Is a Heparin-Binding Site

Enjyoji¹,

Miyata²,

Kamikubo³

et al. 1995

Biochemistry

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“…Several studies of the binding of proteins to heparin have used changes in protein tryptophan fluorescence to monitor binding. Interestingly, in all cases, the tryptophan fluorescence is enhanced upon heparin binding; fluorescence enhancements ranging from 32 to 40% have been reported for the binding of antithrombin III to high molecular weight heparin (Jordan et al, 1980;Olson, 1988; Villanueva & Allen, 1983;Villanueva, 1984;; Blackburn & Sibley, 1980; Bjork & Nordling, 1979; Villanueva et al, 1980). Much larger tryptophan fluorescence enhancements (~4-5-fold) have been reported for heparin binding to mucus proteinase inhibitor (MPI) (Faller et al, 1992).…”

Section: Thermodynamics Of Positively Charged Oligopeptidesmentioning

confidence: 95%

Thermodynamics of Charged Oligopeptide-Heparin Interactions

Mascotti¹,

Lohman²

1995

Biochemistry

101

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To better understand the electrostatic component of the interaction between proteins and the polyanion heparin, we have investigated the thermodynamics of heparin binding to positively charged oligopeptides containing lysine or arginine and tryptophan (KWK-CO2 and RWR-CO2). The binding of these peptides to heparin is accompanied by an enhancement of the peptide tryptophan fluorescence, and we have used this to determine equilibrium binding constants. The extent of fluorescence enhancement is similar for both peptides, suggesting that the tryptophan interaction is similar for both. Titrations of these peptides with a series of simple salts suggest that this fluorescence enhancement is due to the interaction of tryptophan with sulfate moieties on the heparin. Equilibrium association constants, Kobs (M-1), for each peptide binding to heparin were measured as a function of temperature and monovalent salt concentration in the limit of low peptide binding density. At pH 6.0, 25 degrees C, 20 mM KCH3CO2, Kobs = 3.2 (+/- 0.3) x 10(3) M-1 for KWK-CO2 binding, whereas Kobs = 4.5 (+/- 0.5) x 10(3) M-1 for RWR-CO2. However, the dependence of Kobs on KCH3CO2 concentration is the same for both oligopeptides, each of which possesses a net charge of +2 at pH 6.0. The logarithm of Kobs is a linear function of the logarithm of [KCH3CO2] over the range from 12 mM < or = KCH3CO2 < or = 30 mM (pH 6.0, 25 degrees C), with (delta log Kobs/delta log [KCH3CO2]) = -2.0 +/- 0.3, indicating that approximately 2 ions are released per bound peptide upon formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Most researchers believe that a necessary condition for rate acceleration is the binding of heparin to AT [see Rosenberg & Damus (1973), Longas et al (1980), Blackburn & Sibley (1980), Feinman (1979), and references cited therein], although some evidence in the literature suggests that the enzyme might be the target for heparin, at least in the reaction with thrombin [see Smith (1977), Hatton & Regoeczi (1977), Machovich et al (1980), and references cited therein], A major question in current research is whether the binding of heparin to either protein alone is sufficient or whether a ternary heparin-AT-enzyme intermediate is needed for rapid formation of the final, stable enzyme-AT complex. Several groups have presented evidence that such a species is required [see Holmer et al (1979Holmer et al ( , 1981, Pomerantz & Owen (1978), Griffith (1982), and references cited therein], but other studies claim (Jordan et al, 1980) that any heparin-thrombin species f From the Department of Biochemistry, State University of New York Downstate Medical Center, Brooklyn, New York 11203. Received March 1,1983.…”

Section: Articlesmentioning

confidence: 99%

“…Although several methods are currently available for monitoring the reaction, none are ideal. Assays for the loss of enzyme activity do provide a direct measure of the progress of the reaction (Jordan et al, 1980;Hatton & Regoeczi, 1977;Griffith, 1982), but they can be time consuming and somewhat cumbersome.…”

Section: Articlesmentioning

confidence: 99%