Fractionation of heparin by chromatography on a tissue plasminogen activator-Sepharose column.

Abstract

Heparin stimulates the activity of tissue plasminogen activator (t-PA) and binds to t-PA. To study this interaction, a complex between t-PA and N-acetylated heparin was formed and then linked to Sepharose. This procedure selectively links the t-PA to the column because the acetylated heparin has no free amino groups. The procedure also protects the heparin-binding site(s) on the enzyme during coupling to the matrix. The t-PA column separates heparin into two fractions, one with low affinity for t-PA and one with high affinity. Both fractions of heparin effectively accelerate inactivation of thrombin by antithrombin m. However, the fractions differ in their ability to stimulate t-PA: the low-affinity heparin has no effect on the activity of t-PA, whereas the high-affinity heparin enhances this activity. These heparin fractions will be useful in characterizing the biochemical basis and physiological consequences of the heparin-t-PA interaction.Heparin can bind many proteins of biological interest, including antithrombin III (AT-IIl), clotting factors (1), growth factors (2, 3), apolipoproteins (4), fibronectin (5), and plasminogen activators (6)(7)(8). The interaction between heparin and AT-III has been extensively studied. The anticoagulant potency of heparin, due primarily to its binding to AT-III (9, 10), depends on the molecular size of the preparation (11)(12)(13)(14) and a particular polysaccharide sequence (15)(16)(17). This work was aided by fractionation of heparin on columns of immobilized AT-Ill and chemically analyzing the fractions that bound most tightly (15,16,(18)(19)(20)(21)(22).Heparin has been reported to enhance fibrinolysis in vitro and in vivo (23). By using purified components of the fibrinolytic system, heparin has been shown to enhance the activity of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (6,7,24). Whereas commercial heparin binds tightly to both t-PA and Lysplasminogen, low-molecular-weight heparin does not bind t-PA and binds Lys-plasminogen only weakly (25). These low-molecular-weight heparins do not stimulate plasmin formation by t-PA but still retain anticoagulant properties, such as binding to AT-III and acceleration of the inactivation of thrombin. Thus, size of the mucopolysaccharide appears important for its interaction with the fibrinolytic system.To study heparin-t-PA interaction, we sought a way to fractionate heparin on columns of immobilized t-PA. This approach was successful only when t-PA was protected by N-acetylated heparin during the coupling of t-PA to the matrix, similar to the protocol used for purification of AT-III-binding heparins (18). The chromatography has separated heparin into two fractions, one with high affinity for t-PA and the other with low affinity for t-PA. The anticoagulant activities of both fractions are similar. These two fractions should be useful in exploring the biochemical basis and clinical consequences of the heparin-t-PA interaction. EXPERIMENTAL PROCEDURES MaterialsHeparin (calcium salt) wa...

Transforming growth factor‐β1 is a heparin‐binding protein: Identification of putative heparin‐binding regions and isolation of heparins with varying affinity for TGF‐β1

“…AT 111-specific heparin has been isolated by affinity chromatography (Andrade- Gordon and Strickland, 1986;Scully et al, 1988;Kim and Linhardt, 1989;Sudhalter et al, 1989), and results in highly anticoagulant heparin, but does not modulate heparin's effect on cell proliferation (Murrell et al, 1989;Sudhalter et al, 1989) or fibrinolysis (Andrade-Gordon and Strickland, 1986). Other investigators have isolated heparin fractions expressing high affinity to fibronectin (Falcone and Salisbury, 1988), heparin-cofactor I1 (Kim and Linhardt, 1989), tissue plasminogen activator (tPA)(Andrade-Gordon and Strickland, 1990), and acidic FGF (Barzu et al, 1989). In all cases, the fractionation of heparin was conducted with affinity columns bearing the native protein, a strategy that was impractical in the present instance due to the high cost of TGF-p1.…”

Heparin oligosaccharides enhance tissue-type plasminogen activator: a correlation between oligosaccharide length and stimulation of plasminogen activation

“…Stein et al ( 1989) and demonstrated that heparin interacts with the amino-terminal finger domain of t-PA to stimulate the rate of plasmin generation. In addition, Andrade-Gordon and Strickland (1990) fractionated heparin into pools with high affinity and low affinity for t-PA and showed that only the high-affinity fraction enhanced Pg activation, although both fractions retained anticoagulant activity.…”

“…The mechanism of heparin pro-fibrinolytic activity is less well characterized. Recently, Andrade-Gordon and Strickland (1990) proposed that heparin may stimulate plasmin generation by serving as a template for binding both t-PA and Pg, in a manner analogous to that by which heparin binds to both antithrombin III and thrombin to inactivate thrombin. Thus, if binding of heparin to both enzyme and substrate is required, the stimulation of Pg activation may correlate with the size of the heparin oligosaccharide employed in the studies.…”

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

“…Thrombolytic therapy using t-PA and other fibrin-selective agents requires heparin as an adjunct in order to prevent new fibrin formation and rethrombosis (23). On the other hand, experiments in purified systems have shown that heparin enhances plasrninogen activation by t-PA and u-PA, which may increase systemic plasmin generation and decrease plasmin generation on the fibrin clot surface, thus impairing fibrin-specific thrombolysis (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, all latter studies have been performed at sub-physiological ionic strength.…”