Binding, internalization and degradation of heparin and heparin fragments by cultured endothelial cells

We have studied the binding to bovine adrenal capillary endothelial cells cultured in vitro of heparin from different sources (porcine heparin Ep. 152 P, Av.M.W. 15.9 Kd and bovine heparin Ep. 756 P, Av.M.W. 12.9 Kd) and heparin fractions of various molecular weights (low molecular weight heparin, LMW 2123 OP, Av.M.W. 4.5 Kd and very low molecular weight heparin, VLMW 1027/45 OP Av.M.W. 2.1 Kd). The binding was specific for heparin; heparan sulphate showed some competition whereas dextran sulphate and glycosaminoglycans did not. We determined the affinity of heparin and heparin fragments for endothelial cells by means of displacement of bound 3H-labeled heparin in response to increasing concentration of unlabeled compounds. The binding of the different heparin fractions depends on their molecular weights. VLMW 1027/45 OP was unable to bind to the cells, whereas LMW 2123 OP showed an affinity 10 times lower then porcine heparin. Bovine adrenal capillary endothelial cells incubated with unfractionated 3H-labeled heparin selectively bound internalized and degraded high molecular weight heparin fractions, as shown by gel filtration of the 3H-labeled heparin both after binding to the cells and after internalization.     

Inactivation and binding of human plasma kallikrein by antithrombin III and heparin.

Purified human antithrombin III was found to inactivate the esterase, the kininogenase, and the plasminogen activator activity of purified plasma kallikrein. The reaction rate was increased by heparin. Kinetic studies indicated second-order kinetics for the reaction between kallikrein and antithrombin III, and a catalytic effect of heparin. SDS polyacrylamide gel electrophoresis revealed that three new bands were generated during the inactivation of kallikrein by antithrombin III. Heparin did not influence the factor XII-dependent activation of prekallikrein in human plasma, whereas the inactivation of kallikrein elapsed slightly more rapidly in plasma samples where heparin was added. It is concluded that antithrombin III probably is of minor importance for the inactivation of kallikrein in plasma under physiological circumstances.     

Antithrombin BM from human plasma: an antithrombin binding moderately to heparin

A human antithrombin was purified app. 60 fold from Cohn fraction IV, to give a single band of about 70.000 molecular weight in polyacrylamide gel electrophoresis. Compared to the similar antithrombin III, this glycoprotein binds only moderately to porcine heparin (hence its name Antithrombin BM), thus requiring higher heparin concentration for full thrombin inhibitor function, and lower ionic strength for elution from a heparin sepharose column. In these respects it resembles "heparin cofactor A", which is, however, characterized by a substantially larger molecular weight. From AT III, AT BM further differs in its absolute dependency on the presence of heparin(oids) for antithrombin activity, in its more pronounced inhibitory specificity largely restricted to thrombin, and in the absence of substantial immunological crossreaction with antibody to AT III. Based on comparative measurements of antithrombin activity in the presence of different amounts of heparin, up to 40% of the antithrombin activity present in human blood may be attributed to AT BM. The in vivo role of this new inhibitor remains to be elucidated.     

Binding of heparin to human endothelial cell monolayer and extracellular matrix in culture

Endothelial binding of heparin contributes to its local antithrombotic action and catabolism. However, it is uncertain whether heparin may be bound to a damaged or even de-endothelialized vessel surface. Therefore, the binding of 3H-heparin to cultured endothelial cell monolayer and extracellular matrix was studied. The binding reached equilibrium after 4 h. 3H-heparin bound to endothelium could be displaced by unlabelled heparin which competed for about 80% of binding. The binding became saturable when the concentration of heparin exceeded 30-times the therapeutical value. Approximately 6 X 10(11) binding sites for heparin per cm2 of endothelium were calculated. Heparin was bound not only to endothelial cells but also to extracellular matrix, even when it was exposed in the absence of cells. About 40% of binding sites were localized in the extracellular matrix fraction. A similar affinity of binding of 3H-heparin to complete endothelium, endothelial cells and extracellular matrix was estimated /Kd of almost 1 microM/. The binding of heparin to extracellular matrix should be considered in the interpretation of heparin interaction with endothelium.     

Simultaneous determination of plasma prothrombin and antithrombin III (heparin cofactor).

A method for the simultaneous determination of plasma prothrombin and antithrombin III has been developed. It involves activation of a dilute plasma solution with Taipan snake venom and measurement of the heparin-mediated loss of thrombin activity after short incubation. The antithrombin III content is quantitated on the basis of results with known mixtures of normal and antithrombin III-deficient plasmas.     

Decreased binding of heparin to antithrombin following the interaction between antithrombin and thrombin

A complex formed between bovine antithrombin and bovine thrombin was shown to elute from a heparin-agarose column at a lower ionic strength than free antithrombin. Moreover, it was demonstrated that less heparin remained attached to the antithrombin-thrombin complex than to free antithrombin, when mixtures of high-affinity heparin and either complex or antithrombin were separated by gel electrophoresis or gel chromatography under identical conditions. These results suggest that the interaction between antithrombin and thrombin leads to a decreased affinity of heparin for the antithrombin moiety of the complex. This decrease may be an essential feature of the proposed function of heparin as a catalyst in the antithrombin-thrombin reaction.     

Reactivity of heparin with the human plasma heparin-binding proteins thrombin, antithrombin III, and apolipoproteins E and B-100

The heparin-binding properties of human plasma apolipoproteins B-100 and E (apoB-100 and E) of low density lipoproteins (LDL), thrombin, and antithrombin-III (AT-III) were investigated. A highly reactive heparin (HRH) to apoB-100 was isolated by chromatography of crude heparin on a column of LDL immobilized to Affi-Gel 10. This HRH showed a high, Ca²⁺-dependent precipitating activity towards LDL; 1 μg HRH uronic acid precipitated 50–70 μg LDL-protein. HRH was fractionated further by chromatography on a column of AT-III bound to concanavalin A-Sepharose. The unretained fraction of heparin (HRH₁)had a low affinity for AT-III. The bound heparin (HRH₂) had a high affinity for AT-III and precipitated LDL in the presence of Ca²⁺. To assess further their heparin-binding properties, the proteins were subjected to gradient-gel electrophoresis under denaturing conditions, transferred to nitrocellulose by electrophoresis, and then assayed for their ability to bind [¹²⁵I]-labeled HRH₂. Autoradiographic analysis showed that thrombin, apolipoproteins E and B-100, and the AT-III-thrombin covalent complex bound HRH₂. Denatured AT-III did not bind HRH₂, indicating that its heparin recognition site may depend on conformation.     

Interaction of thrombin with heparin cofactor II and antithrombin III on prostacyclin production by cultured endothelial cells

To investigate the physiologic function of heparin cofactor II (HCII), endothelial cells from human umbilical vein were incubated in vitro for 20 min with 0.5 NIH U/ml thrombin in the presence of HCII or antithrombin III (ATIII), and prostacyclin production determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Although ATIII at 20 mInh.U/ml slightly but significantly inhibited thrombin-induced prostacyclin production, neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) at 1 U/ml accelerated the inhibitory effect of ATIII. HCII at 10 and 20 mInh.U/ml did not decrease thrombin stimulation of prostacyclin production in the presence or absence of UFH or LMWH. However, HCII caused a marked decrease in the thrombin-stimulated prostacyclin prostacyclin production in the presence of 2 mg/ml dermatan sulfate (DS). The significant inhibition by HCII occurred when the DS concentrations were 0.2 microgram/ml and higher. From these results we suggest that HCII may prevent a prostacyclin-induced inhibition of platelet aggregation for hemostasis when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of DS.     

Binding of heparin on the surface of cultured human endothelial cells

³H-labelled heparin was shown to bind to the surface of cultured human endothelial cells. The binding was time dependent, saturable, reversible and trypsin sensitive. The binding capacity corresponds to about 10⁶ molecules of heparin per cell. Labelled material released from the cells retained its ability to bind to antithrombin III. Binding of heparin to endothelial cells as well as its release is suggested to be of importance in the pharmacokinetics of heparin and to influence the non-thrombogenic properties of the endothelial lining during heparin therapy.     

Enhancement by heparin of thrombin-induced antithrombin III proteolysis: Its relation to the molecular weight and anticoagulant activity of heparin

Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. We now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of α-thrombin with a molar excess of ¹²⁵I AT III. Proteolysis of the inhibitor was assessed by SDS polyacreylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistant with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.     

Contact activation of blood coagulation is inhibited by plasma factor XIII b-chain

The effect of the purified bovine plasma factor XIII b-chain on contact activation of blood coagulation was studied in human and bovine plasma using either an ellagic acid-phospholipid suspension (Cephotest) or dextran sulfate as activating surface. Contact activation was monitored by the generation of amidolytic activity towards a synthetic chromogenic substrate (S-2302) for factor XIIa and plasma kallikrein. The factor XIII b-chain, which is released from tetrameric factor XIII (a2b2) in the late stages of blood coagulation, inhibits contact activation induced by both activation surfaces mentioned. It was shown that a 5 min preincubation of the factor XIII b-chain with the activation surface increases its inhibitory effect. Light scattering measurements indicated a concurrent binding of the factor XIII b-chain to the Cephotest material. Because factor XIII (a2b2) itself had no such inhibitory activity, the present finding that the factor XIII b-chain inhibits contact activation may point to a novel feed-back inhibition mechanism of blood coagulation.     

Competitive adsorption of plasma proteins onto polymer surfaces

Competitive adsorption of plasma proteins onto polymer surfaces has been investigated by using I-125 radiolabelled proteins to determine the rate and adsorbed concentration of each protein from a mixture of albumin, γ-globulin and fibrinogen in a phosphate buffered saline solution. Fibrinogen adsorbed the fastest on all the polymers studied but each protein adsorbed with a rate and concentration dependent upon both the polymer and the protein. The results allow some predictions concerning events occurring when blood contacts polymer surfaces.     

Separation of active and inactive forms of human antithrombin by heparin affinity chromatography

During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human alpha-antithrombin, human beta-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 degrees C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the alpha-latent and beta-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.     

Modification of an amidolytic heparin assay to express protein-bound heparin and to correct for the effect of antithrombin III concentration

A modification of an anti-Xa assay for plasma heparin has been devised using a low molecular weight dextran sulfate that competitively binds protein heparin neutralizers and displaces masked heparin. The addition of 0.12 mg dextran sulfate per ml of plasma permits heparin, neutralized by the products of platelet aggregation, to recover full functional activity against Xa. The assay will permit a more accurate assessment of both exogenous plasma heparin and endogenous heparin-like activity in blood samples collected with varying techniques. A further modification is proposed employing polybrene to neutralize the plasma heparin-like material providing a concurrent control for each sample that increases accuracy by eliminating the effect of varying AT-III levels which have anti-Xa activity.