Alan R. Stafford

HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulant activity.

The appearance of anionic lipids on the extracellular surface of cells is required for the formation of the procoagulant complex that leads to the activation of prothrombin. Procoagulant activity would be expected to be inhibited by substances that stabilize the membrane structure and hence inhibit the transbilayer diffusion of phosphatidylserine from the cytoplasmic to the extracellular surface of the plasma membrane. The generation of procoagulant activity in human erythrocytes by A23187 and Ca2+ is inhibited by apolipoprotein A-I, its amphipathic peptide analogues, and high-density lipoprotein (HDL). These agents do not inhibit the Ca2+ loading of erythrocytes by A23187, nor do they inhibit the activation of prothrombin once the cells have been incubated at 37 degrees C with A23187 and Ca2+. Transbilayer diffusion of fluorescently labeled phosphatidylserine is inhibited by apolipoprotein A-I. These findings indicate that class A amphipathic helixes as well as lipoprotein particles and liposomes inhibit the transbilayer diffusion of phospholipids and procoagulant activity. This activity may contribute to the protective role of HDL against arteriosclerosis and thrombosis.

Evidence for Allosteric Linkage between Exosites 1 and 2 of Thrombin

Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH2Cl (FPR)-thrombin. Hirudin-(54–65) and sF2, a synthetic peptide corresponding to residues 63–116 of prothrombin fragment 2, were used as ligands for exosites 1 and 2 of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPR-thrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF2 and hirudin-(54–65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF2 is titrated with hirudin-(54–65), complete displacement of fluorescein-sF2 is observed. Likewise, when thrombin-bound fluorescein-hirudin-(54–65) is titrated with sF2, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.

Exosites 1 and 2 Are Essential for Protection of Fibrin-bound Thrombin from Heparin-catalyzed Inhibition by Antithrombin and Heparin Cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (γ-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 → Ala, Arg97 → Ala, and Arg101 → Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, α1-antitrypsin Met358 → Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.

Increased accumulation of drugs in a multidrug resistant cell line by alteration of membrane biophysical properties

Growth of CHRC5 multidrug resistant cells in media enriched in a saturated C-17 fatty acid, heptadecanoic acid, resulted in these cells accumulating vinblastine at a rate and to an extent comparable to that of the parental cell line AB1. The fatty acid-enriched growth media had no effect on the ability of AB1 cells to take up vinblastine. The action of amphiphiles on the uptake of rhodamine dyes by CHRC5 cells was compared with the increased dye accumulation affected by verapamil. Membrane rigidifying agents, such as the saturated fatty acid stearic acid, or the cholesterol derivatives, cholesteryl hemisuccinate and cholesteryl phosphorylcholine, as well as a membrane fluidizing unsaturated fatty acid, linoleic acid, could significantly increase dye uptake, although not as well as verapamil. These results taken in conjunction with other reports in the literature, demonstrate that multidrug resistance is sensitive to alterations of membrane properties. They suggest that perturbation of the membrane to either increased or to decreased membrane fluidity can lower the level of resistance.

Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits.

Central venous catheter thrombosis can cause venous obstruction and pulmonary embolism. To determine the extent to which catheter thrombosis is triggered by the contact or extrinsic pathway of coagulation, we used antisense oligonucleotides (ASOs) to selectively knock down factor (f)XII, fXI, or high-molecular-weight kininogen (HK), key components of the contact pathway, or fVII, which is essential for the extrinsic pathway. Knockdown of contact pathway components prolonged the activated partial thromboplastin time and decreased target protein activity levels by over 90%, whereas fVII knockdown prolonged the prothrombin time and reduced fVII activity to a similar extent. Using a rabbit model of catheter thrombosis, catheters implanted in the jugular vein were assessed daily until they occluded, up to a maximum of 35 days. Compared with control, fXII and fXI ASO treatment prolonged the time to catheter occlusion by 2.2- and 2.3-fold, respectively. In contrast, both HK and fVII knockdown did not significantly prolong the time to occlusion, and dual treatment with fVII- and fXI-directed ASOs produced a time to occlusion similar to that with the fXI ASO alone. These findings suggest that catheter thrombosis is triggered via the contact pathway and identify fXII and fXI as potential targets to attenuate this complication.

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg184, Lys185, Arg189, Arg192, Arg193, demonstrated a ∼130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates γ-thrombin at a 65-fold slower rate than α-thrombin, (b) hirudin-(54–65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation ∼100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.

Mechanism of catheter thrombosis: comparison of the antithrombotic activities of fondaparinux, enoxaparin, and heparin in vitro and in vivo

In patients undergoing percutaneous coronary intervention, catheter thrombosis is more frequent with fondaparinux than heparin. This study was undertaken to identify the responsible mechanism and to develop strategies for its prevention. Percutaneous coronary intervention catheter segments shortened plasma clotting times from 971 ± 92 to 352 ± 22 seconds. This activity is factor XII (fXII) dependent because it was attenuated with corn trypsin inhibitor and was abolished in fXII-deficient plasma. Heparin and enoxaparin blocked catheter-induced clotting at 0.5 and 2 anti-Xa U/mL, respectively, whereas fondaparinux had no effect. Addition of fondaparinux to bivalirudin or low-dose heparin attenuated catheter-induced clotting more than either agent alone. In a rabbit model of catheter thrombosis, a 70 anti-Xa U/kg intravenous bolus of heparin or enoxaparin prolonged the time to catheter occlusion by 4.6- and 2.5-fold, respectively, compared with saline, whereas the same dose of fondaparinux had no effect. Although 15 anti-Xa U/kg heparin had no effect on its own, when given in conjunction with 70 anti-Xa U/kg fondaparinux, the time to catheter occlusion was prolonged 2.9-fold. These findings indicate that (1) catheters are prothrombotic because they trigger fXII activation, and (2) fondaparinux does not prevent catheter-induced clotting unless supplemented with low-dose heparin or bivalirudin.

Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Thrombin exosite 1 binds the predominant γA/γA-fibrin form with low affinity. A subpopulation of fibrin molecules, γA/γ′-fibrin, has an extended COOH terminus γ′-chain that binds exosite 2 of thrombin. Bivalent binding to γA/γ′-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from γA/γ′-fibrin clots than from γA/γA-fibrin clots. After 24 h of washing, however, both γA/γ′- and γA/γA-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in γA/γ′-fibrin clots than they were in γA/γA-fibrin clots. This difference reflects bivalent binding of thrombin to γA/γ′-fibrin because (a) it is abolished by addition of a γ′-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the γ′-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with γA/γ′-fibrin than with γA/γA-fibrin clots. Thus, bivalent binding of thrombin to γA/γ′-fibrin protects thrombin from inhibition, raising the possibility that γA/γ′-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Dimerization of the P-glycoprotein in membranes.

Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis. The P-glycoprotein content of the membrane was determined by Western blots. Irradiation resulted in the loss of P-glycoprotein. The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein. This is strong evidence to indicate that the P-glycoprotein self associates in the membrane.

Long Range Communication between Exosites 1 and 2 Modulates Thrombin Function

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), γ′-peptide (an analog of the COOH terminus of the γ′-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to γA/γA-fibrin, an interaction mediated solely by exosite 1. When γA/γA-fibrinogen was clotted with thrombin in the presence of HD22, γ′-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for γA/γA-fibrin 6-fold and accelerated the dissociation of thrombin from preformed γA/γA-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with γA/γA-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54–65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized γ′-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.