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%.     

The effects of heparin and low molecular weight heparins on bone

Recent clinical trials have shown that the risk of developing osteoporosis is substantially lower when low molecular weight heparins (LMWHs) are used in place of unfractionated heparin. While the reason(s) for this difference has not been fully elucidated, studies with animals have suggested that heparin causes bone loss by both decreasing bone formation and increasing bone resorption. In contrast, LMWHs appear to cause less bone loss because they only decrease bone formation. Whether all LMWHs decrease bone formation and therefore cause bone loss is unknown. For example, preliminary in vitro studies with the synthetic pentasaccaride, Fondaparinux, have suggested that it may not decrease bone formation and thus, may have no deleterious effects on bone. Further studies are required in order to determine if all LMWHs cause bone loss equally.     

Purification of thrombin by affinity chromatography on immobilized heparin

Bovine thrombin has been purified from commercial crude thrombin preparations by affinity chromatography on immobilized heparin. This procedure was shown to be superior to affinity chromatography on matrix-linked synthetic low-molecular-weight thrombin inhibitors in that the purified enzyme could be conveniently eluted by a salt gradient and also was of higher purity. The degree of purification of the affinity chromatography step was 30 times and the purified material had a specific activity of about 2000 NIH units/mg. Dodecyl sulphate-polyacrylamide gel electrophoresis indicated the presence of the native two-chain form of thrombin as well as of several forms of thrombin with specific proteolytic cleavages in the B-chain. These modified forms were similar to those also found in thrombin preparations obtained from crude commercial thrombin by other methods. Affinity chromatography on heparin-agarose was shown to be useful also for the purification of thrombin from a prothrombin activation mixture.     

Factors influencing the separation of glu-plasminogen affinity forms I and II by affinity chromatography

Native human plasminogen, the proenzyme of plasmin (E.C. 3.4.21.7) occurs in blood in two well defined forms, affinity forms I and II. In this paper, the feasibility of separating these forms of human native plasminogen by affinity chromatography, is shown to be dependent on two factors: 1) the ionic composition of the buffer containing the displacing agent: buffers of varying contents of sodium, Tris, phosphate and chloride ions were compared, and 2) the type of adsorbent. Two adsorbents were compared: Sepharose-lysine and Sepharose-bisoxirane-lysine. Only in the phosphate containing buffers, irrespective of the type of adsorbent, the affinity forms can be separated. The influence of the adsorbent can be accounted for by a large difference in dissociation constants of the complex between plasminogen and the immobilized lysine.     

Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Heparin purified by affinity chromatography on anti-thrombin III-Sepharose has been studied by various methods. The specific activities of the materials obtained were in the range 170–230 units/mg as determined by a whole plasma clotting method and 360–780 units/mg as determined by a F.X_a inhibition method. Gel filtration of the material showed that there was a definite molecular size dependency of the specific activities and the activity profiles were markedly different when assay -ed by different methods. These features were also observed (at generally lower activities) with gel filtration fractions of commercial heparin. The possible conclusions regarding the mechanism of heparin anticoagulant action are discussed.     

Co-administration of low molecular weight heparin enhances the profibrinolytic effect of warfarin through different mechanisms

Background and Objective

Treatment with vitamin K antagonists (VKA) reduces fibrinolytic resistance through the inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor (TAFI). Because low-molecular weight heparin (LMWH) is co-administered with VKA during initiation of anticoagulant treatment, we evaluated the effect of dual anticoagulation on fibrinolytic resistance.

Patients and Methods

Two groups of patients were studied: 1) patients on stable warfarin; 2) patients starting oral anticoagulant therapy, who were evaluated during dual anticoagulation and after enoxaparin withdrawal. Only samples with an INR between 2 and 3 were compared. The resistance of clots to t-PA-induced fibrinolysis was evaluated in blood and plasma by thromboelastography (TEG) and turbidimetry, respectively.

Results

In patients on dual anticoagulation, blood fibrinolysis time (TEG) was significantly shorter than in patients on warfarin alone and significantly correlated with LMWH level. The profibrinolytic effect was partly ascribable to a reduction of thrombin-dependent TAFI activation: 1) thrombin and TAFIa generation were significantly reduced by dual anticoagulation; 2) the addition of enoxaparin to warfarin-blood reduced TAFI-mediated fibrinolysis inhibition. Patients on dual anticoagulation also displayed a reduction in clot strength, a phenomenon known to reduce fibrinolytic resistance. The profibrinolytic effect of LMWH co-administration was not seen in plasma, likely because TAFIa generation was below the threshold required to inhibit fibrinolysis.

Conclusions

Co-administration of LMWH in patients under VKA reduces the fibrinolytic resistance of blood clots via TAFI-dependent and TAFI-independent mechanisms. Further studies are warranted to assess the clinical implications of these findings.     

Differential activities of heparins in human plasma and in sheep plasma. Effects of heparin molecular sizes and sources

We analyzed the anticoagulant activity of heparins in human plasma and in sheep plasma upon recalcification using the technique of clot elasticity measurement. Heparins with different molecular weight (M.W.) distributions, fractionated by Ultrogel AcA 44 gel chromatography, and heparins from different sources were analyzed in these two coagulation systems. In both systems, heparin with M.W. approximating 20,000 showed the highest specific activity; activities dropped with either increasing or decreasing M.W. Activities of each heparin fraction in these two clotting systems were not parallel. The differences were strongly dependent on the M.W. and sources. The activity-M.W. curves determined in these two clotting systems crossed at a position with M.W. 17,000, a value in the middle of the M.W. range of most commercial heparins (approximately 12,000 to 20,000). This indicates that the United States Pharmacopoeia (USP) method, which uses sheep plasma, underestimates the anticoagulant activity of heparins in human plasma with M.W. between 17,000 and 25,000 and overestimates that with M.W. lower than 17,000. The greatest discrepancy was found with the smallest M.W. fraction (6,200); an 800% difference was observed. Discrepancies were also observed with heparins from different sources. The USP method using porcine mucosal standard underestimates the potency of a gut heparin in human plasma by a factor of 1.5, and overestimates potencies of heparins from beef origin, either mucosal or lung material, by approximately 20%. A heparin preparation isolated from whale showed no discrepancy.     

Cellular and molecular mechanisms in the long-term action of antidepressants.

The hypotheses on the pathophysiology of depression /mood disorders and on antidepressant mechanisms have greatly changed in recent years. The classical monoamine hypothesis was revealed to be simplistic, in that it could not explain the temporal delay in the therapeutic action of antidepressants. Converging lines of evidence have shown that adaptive changes in the several mechanisms of neuroplasticity are likely to be the cellular and molecular correlates of therapeutic effect. In this article, several mechanisms of neuroplasticity are analyzed in relation to the mechanism of antidepressants, ranging from changes in gene expression (including neurotrophic mechanisms), to synaptic transmission and plasticity, and neurogenesis. We propose that the current version of the hypothesis of antidepressant mechanism simply be called the “hypothesis of neuroplasticity. ” In the final section, we also briefly review the main current novel strategies in the pharmacology of depression and the new putative targets for antidepressants, with particular emphasis on nonmonoaminergic mechanisms.     

Endothelial capillary tube formation and cell proliferation induced by tumor cells are affected by low molecular weight heparins and unfractionated heparin

BACKGROUND: Clinical studies suggest a survival advantage in cancer patients receiving low molecular weight heparin (LMWH). A suggested mechanism for this beneficial effect may reside in the antiangiogenic activity of heparins. OBJECTIVES: In this study we investigated whether two different LMWHs, i.e. enoxaparin and dalteparin, and unfractionated heparin (UFH), affect the angiogenic potential of human microvascular endothelial cells (HMEC-1) promoted by tumor cells. METHODS: HMEC-1 cells were incubated with tumor cell conditioned media (TCM) derived from human breast cancer and leukemic cells (i.e. MCF-7, MDA.MB.231, and NB4 cell lines) or recombinant cytokines (i.e. VEGF, FGF-2, TNF-alpha) +/-heparins. Capillary-like tube formation in Matrigel and cell proliferation were evaluated. RESULTS: All three TCM induced a significant (p<0.05) increase in total length of tubes formed by HMEC-1 in Matrigel. These increases were significantly counteracted (62 to 100% mean inhibition) by enoxaparin and dalteparin, but were significantly less affected by UFH. Similarly, the tube formation induced by standard VEGF, FGF-2, or TNF-alpha was 100% inhibited by enoxaparin, and 70-90% by dalteparin, whereas minor or no inhibition was observed with UFH. VEGF was the most active cytokine in TCM of both breast cancer and leukemic cells. EC proliferation was significantly increased by standard angiogenic factors, and slightly affected by breast cancer TCM (p=ns). The addition of heparins significantly counteracted the proliferative stimuli. CONCLUSIONS: These results support a major role for LMWH compared to UFH in inhibiting the proangiogenic effect exerted by tumor cells or purified angiogenic factors on microvascular endothelium.     

Adverse effect of heparin on antithrombin action during endotoxemia: microhemodynamic and cellular mechanisms

A recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin + AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH + AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPS-treated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p < 0.01), whereas Heparin + AT and LMWH + AT animals showed microcirculatory alterations comparable to that in controls. In additional in vitro chemotaxis assays. AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH. Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia. These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.     

Migraine: new molecular mechanisms.

Migraine is an episodic headache disorder affecting more than 10% of the general population. Migraine arises from a primary brain dysfunction that leads to activation and sensitization of the trigeminovascular system. A major incompletely understood issue in the neurobiology of migraine concerns the molecular and cellular mechanisms that underlie the primary brain dysfunction and lead to activation and sensitization of the trigeminovascular system, thus generating and maintaining migraine pain. Here the author reviews recent discoveries that have advanced our understanding of these mechanisms toward a unifying pathophysiological hypothesis, in which cortical spreading depression (CSD), the phenomenon underlying migraine aura, assumes a key role. In particular, the author discusses the main recent findings in the genetics and neurobiology of familial hemiplegic migraine and the insights they provide into the molecular and cellular mechanisms that may lead to the increased susceptibility of CSD in migraineurs.     

Purification of antithrombin III by affinity chromatography

Human antithrombin III has been purified from plasma by affinity chromatography on heparin-Sepharose gel. After further fractionation on DEAE-Sephadex and Sephadex G 200 a homogeneous preparation of molecular weight 67 000 was obtained. Amino acid analysis showed that cystine and cysteine were absent. The carbohydrate content was found to be 15.2 %, and N-terminal analysis gave only histidine. Antithrombin III presumably consists of a single polypeptide chain without disulphide bridges. The pure preparation showed both progressive antithrombin activity and heparin cofactor activity.     

Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute to the action of this drug in blood coagulation.     

Standardization of low molecular weight heparins: a collaborative study

A collaborative study was carried out, in which eight laboratories each assayed eight low molecular weight (LMW) heparins against the International Standard (IS) for heparin. APTT assays and three types of anti-Xa method were used. The results of this study showed that: LMW heparins cannot be validly assayed against the IS by APTT or anti-Xa methods. Potencies of LMW heparins vs. the IS differed considerably between the four types of assay method used and also between different laboratories using the same type of method. Adoption of a single LMW heparin standard would improve validity, improve inter-laboratory variation, and largely abolish the differences between the three types of anti-Xa method. However, since calibration of a LMW heparin standard against the IS would give potencies that differ widely by the different assay methods, a single assay method such as the anti-Xa amidolytic, plasma, would need to be chosen for this calibration.     

Study of low molecular weight heparin effect on the relation between anticoagulant activity and antithrombin III affinity.

Low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) were fractionated by rabbit antithrombin III (AT III)-Sepharose, and the effects of each affinity fraction on the coagulation and fibrinolytic activities were investigated. FR-860 was fractionated to no-affinity, low-affinity (LA) and high-affinity (HA) fractions, and UF-heparin to LA and HA fractions. The HA fractions showed higher activities regarding the prolongation of activated partial thromboplastin time, anti-factor Xa activity and antithrombin activity compared with those of LA. The HA and LA fractions exhibited the enhancement of heparin cofactor II (HC II) activity and fibrinolytic activity in a dose-dependent manner. These results suggest that the antithrombotic activity of FR-860 is exerted through AT III and other mechanism such as HC II-mediated system.     

The effect of heparin fragments of different molecular weights on experimental thrombosis and haemostasis

The effect of heparin fragments of different molecular weights has been compared with that of conventional sodium heparin on experimental thrombosis in vivo and ex vivo and experimental haemostasis in vivo. In the first part of the study fragments of different molecular weights were given (4,900, 6,500, 9,500 and 22,200 dalton). All preparations including the control gave a significant prolongation of the haemostatic plug formation time in the rabbit mesenteric microcirculation, and all except the fragment with the lowest molecular weight reduced the frequency of jugular vein thrombosis (induced by a combination of endothelial denudation and stasis). There was a correlation between the XaI activity of the different heparin fragments and frequency of thrombosis. Using an ex vivo method (modification of Chandler's model) a dose dependent lag phase until start of thrombus formation was found. In the second part of the study a dose response investigation was made comparing different doses of a fragment (6,500 dalton) with conventional heparin in the same XaI doses (10, 30 and 60 units/kg). Sodium heparin in the highest dose prolonged the haemostatic plug formation time whereas none of the fragment doses did. The lowest dose both of the fragment and conventional heparin did not reduce the frequency of thrombosis, whereas the two higher doses did. Thus it may be possible to obtain preventive effect on thrombus formation with a heparin fragment.