Determination of molecular weight of heparin by size exclusion chromatography with universal calibration

The molecular weight (MW) of heparin can be accurately determined by size exclusion chromatography using "universal calibration." A universal calibration curve was constructed for Superose 12 with standard pullulan samples and verified using characterized ficoll fractions. This calibration yielded the correct MW of heparin as determined by light scattering, when the ionic strength of the mobile phase was maintained over 1.0M. Sodium poly(styrenesulfonate) samples were not suitable standards because of adsorption at high salt concentration and repulsion from the packing material at low ionic strength. The extraordinarily high charge density of heparin leads to the need for high salt concentration to screen such repulsions.     

Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides

Heparin was divided into four fractions on fibronectin-Sepharose. The higher affinity fraction for fibronectin was larger in molecular size, higher in sulfate content and higher in affinity for anti-thrombin III. Together with these heparin fractions, the following three series of heparin samples were examined to compare the affinity for fibronectin-Sepharose: four fractions separated on Sephadex G-100; five fractions separated on antithrombin III-Sepharose, and six partially and completely N-desulfated heparins. The result showed that the affinity of heparin for fibronectin was dependent exclusively on its molecular size, and that an appropriate level of sulfate content in heparin (1.9-2.4 mol/disaccharide) was essential for the affinity. The sulfated preparations of glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin 4-sulfate) and neutral polysaccharides (amylose and dextran) having higher sulfate content than heparin were found to display higher affinity for fibronectin than heparin. This suggested that highly sulfated polysaccharides showed potent affinity irrespective of their polysaccharide structure. The sulfated chondroitin 4-sulfate having a sulfate content and molecular size comparable to those of heparin was inferior to heparin with respect to affinity. A competitive dissociation experiment indicated that heparin and other polysulfated polysaccharides share a common binding site on the fibronectin molecule.     

The molecular-weight range of mucosal-heparin preparations.

A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.     

Structural basis for the anticoagulant activity of heparin. 1. Relationship to the number of charged groups.

This study was undertaken to provide further information concerning the chemical heterogeneity of heparins and the relationships between the anticoagulant activity (USP assay) and the anionic density of the heparin. A sample of commercial heparin was fractionated into 13 fractions by sequential extraction in a two-phase system of 1-butanol-aqueous NaCl containing excess hexadecylpyridinium chloride. The anionic density distribution was characterized by the fractional distribution of uronate among the fractions. The fractions were characterized by several molar ratios of constituents, molecular weight, charge density, and anticoagulant activity in recalcified sheep plasma. The heparin was broadly distributed among the last 10 fractions; the first three contained impurities which were completely separated from the heparin fractions. The heparin fractions differ systematically in anionic density but are of substantially the same molecular weight. Anticoagulant activity increased markedly with anionic density, ranging from 81 units/mg for the heparin fraction with the lowest anionic density up to a high of 243 units/mg. The relationship between anticoagulant activity and either anionic density or its square is nonlinear. However, in the latter case an initial linear relationship was observed for anticoagulant activities of less than 200 units/mg.     

Structural and activity variability of fractions with different charge density and chain length from pharmaceutical heparins

Heparin is a structurally complex polysaccharide used as a clinical anticoagulant. It is comprised of a heterogeneous mixture of polysaccharide chains having a variety of sequences and lengths. The production methods and regulatory controls of pharmaceutical heparins have changed over the years. This study assesses the structural and activity uniformity of the polysaccharide chains comprising two contemporary heparin products. The heparin fractions with different sizes and charges were separated with size exclusion and ion exchange chromatography. The fractions were analyzed for their molecular weight properties, di- and tetrasaccharide compositions, and anti-factor IIa and anti-factor-Xa activities. The distribution of these properties through chains of different lengths and ones with different charge density were compared. The results demonstrate that with the increase in heparin purity, activity and molecular weight required by the current pharmacopeia, the uniformity of pharmaceutical heparin products have increased.     

Small-angle x-ray scattering of high- and low-affinity heparin

Solution characterization of heparin with high affinity (HA) and low affinity (LA) for antithrombin III was performed using the methods of small-angle x-ray scattering (SAXS), viscometry, and aqueous gel permeation chromatography (GPC). SAXS provided various topological parameters including the radius of gyration ([S2]1/2), radius of gyration of cross-section ([S2]q1/2), persistence length (a*), contour length (L), and mass parameters, e.g., overall molecular mass (Mr), and mass per unit length (Mq). The molecular weights of HA and LA pig mucosal heparins were found to be 14,900 and 11,500 and the respective radii of gyration were 40.1 and 33.6 A. The persistence lengths of HA and LA were 21.3 and 20.3 A, respectively. These parameters were compared to SAXS data of heparin [S. S. Stivala, M. Herbst, O. Kratky, and I. Pilz (1968) Arch. Biochem. Biophys. 127, 795-802] fractionated according to molecular weight only. It was found that the various experimental values of this heparin lie somewhere in between those of HA and LA heparins. It appears that there are no appreciable differences in the physico-chemical properties, including conformation, among the heparins in H2O at 25 degrees C.     

Kinetic analysis of various heparin fractions and heparin substitutes in the thrombin inhibition reaction

Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction.     

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.     

Inhibition of allergic airway responses by inhaled low-molecular-weight heparins: molecular-weight dependence

Martinez-Salas, José, Richard Mendelssohn, William M. Abraham, Bernard Hsiao, and Tahir Ahmed. Inhibition of allergic airway responses by inhaled low-molecular-weight heparins:molecular-weight dependence. J. Appl.Physiol. 84(1): 222-228, 1998.Inhaled heparin prevents antigen-induced bronchoconstriction and inhibitsanti-immunoglobulin E-mediated mast cell degranulation. We hypothesizedthat the antiallergic action of heparin may be molecular weightdependent. Therefore, we studied the effects of three differentlow-molecular-weight fractions of heparin [medium-, low-, andultralow-molecular-weight heparin (MMWH, LMWH, ULMWH,respectively)] on the antigen-induced acute bronchoconstrictorresponse (ABR) and airway hyperresponsiveness (AHR) in allergic sheep.Specific lung resistance was measured in 22 sheep before and afterairway challenge with Ascarissuum antigen, without and afterpretreatment with inhaled fractionated heparins at doses of0.31-5.0 mg/kg. Airway responsiveness was estimated before and 2 hpostantigen as the cumulative provocating dose of carbachol in breathunits that increased specific lung resistance by 400%. Allfractionated heparins caused a dose-dependent inhibition of ABR andAHR. ULMWH was the most effective fraction, with the inhibitory dosecausing 50% protection (ID₅₀)against ABR of 0.5 mg/kg, whereasID₅₀ values of LMWH and MMWH were1.25 and 1.8 mg/kg, respectively. ULMWH was also the most effective fraction in attenuating AHR; theID₅₀ values for ULMWH, LMWH, andMMWH were 0.5, 2.5, and 4.7 mg/kg, respectively. These data suggestthat 1) fractionatedlow-molecular-weight heparins attenuate antigen-induced ABR and AHR;2) there is an inverse relationship between the antiallergic activity of heparin fractions and molecular weight; and 3) ULMWH is the mosteffective fraction preventing allergic bronchoconstriction and airwayhyperresponsiveness.

     

Low molecular weight heparin in prevention of perioperative thrombosis.

OBJECTIVE--To determine whether prophylactic treatment with low molecular weight heparin reduces the incidence of thrombosis in patients who have had general or orthopaedic surgery. DESIGN--Meta-analysis of results from 52 randomised, controlled clinical studies (29 in general surgery and 23 in orthopaedic surgery) in which low molecular weight heparin was compared with placebo, dextran, or unfractionated heparin. SUBJECTS--Patients who had had general or orthopaedic surgery. INTERVENTION--Once daily injection of a low molecular weight heparin compared with placebo, dextran, or unfractionated heparin. MAIN OUTCOME MEASURES--Incidence of deep venous thrombosis, pulmonary embolism, major haemorrhages, and death. RESULTS--The results confirm that low molecular weight heparins are more efficacious for the prophylactic treatment of deep venous thrombosis than placebo (common odds ratio 0.31, 95% confidence interval 0.22 to 0.43; p < 0.001) and dextran (0.44, 0.30 to 0.65; p < 0.001). The results suggest that low molecular weight heparins are also more efficacious than unfractionated heparin (0.85, 0.74 to 0.97; p = 0.02), with no significant difference in the incidence of major haemorrhages (1.06, 0.93 to 1.20; p = 0.62). CONCLUSIONS--Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. However, it remains to be shown in a suitably powered clinical trial whether low molecular weight heparin reduces the risk of fatal pulmonary embolism compared with heparin.     

Electrofocusing of heparin: fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides.

Twenty-one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular-weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N- and O-desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular-weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.     

Comparison of efficacy and safety of low molecular weight heparins and unfractionated heparin in initial treatment of deep venous thrombosis: a meta-analysis.

OBJECTIVE--To compare the efficacy and safety of low molecular weight heparins and unfractionated heparin in the initial treatment of deep venous thrombosis for the reduction of recurrent thromboembolic events, death, extension of thrombus, and haemorrhages. DESIGN--Meta-analysis of results from 16 randomised controlled clinical studies. SUBJECTS--2045 patients with established deep venous thrombosis. INTERVENTION--Treatment with low molecular weight heparins or unfractionated heparin. MAIN OUTCOME MEASURES--Incidences of thromboembolic events (deep venous thrombosis or pulmonary embolism, or both); major haemorrhages; total mortality; and extension of thrombus. RESULTS--A significant reduction in the incidence of thrombus extension (common odds ratio 0.51, 95% confidence interval 0.32 to 0.83; P = 0.006) in favour of low molecular weight heparin was observed. Non-significant trends also in favour of the low molecular weight heparins were observed for the recurrence of thromboembolic events (0.66, 0.41 to 1.07; P = 0.09), major haemorrhages (0.65, 0.36 to 1.16; P = 0.15), and total mortality (0.72, 0.46 to 1.4; P = 0.16). CONCLUSIONS--Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in the treatment of venous thrombosis. These results, however, remain to be confirmed by using clinical outcomes in suitably powered clinical trials.     

Hydrophobic-interaction chromatography of glycosaminoglycuronans: the contribution of N-acetyl groups in heparin and heparan sulfate to the affinity for hydrophobic gels, and variety of molecular species in beef-kidney heparan sulfate

Contribution of N-acetyl groups in heparin and heparan sulfate to their affinity for hydrophobic gels was examined by use of a series of semi-synthetic, N-acetylated, hog-intestinal heparins, a whale-intestinal heparin, and a beef-kidney heparan sulfate. Chromatography on Phenyl-Sepharose CL-4B in 3.8-1.0M ammonium sulfate-10mM hydrochloric acid indicated that an increasing N-acetyl content, which is correlated to a decreasing N-sulfate content, results in a marked increase in the affinity for the gels. The variety of molecular species in beef-kidney heparan sulfate, previously fractionated by conventional chromatographic procedures, was demonstrated by separating further, by hydrophobic-interaction chromatography, the polysaccharide into several fractions composed of molecular species distinctly different in N-acetyl and sulfate content, and in molecular size.     

Dynamic light scattering studies of porcine submaxillary mucin fractions in dilute solution at intermediate scattering vectors

We report dynamic light scattering measurements over a wide range of scattering vectors for fractionated samples of porcine submaxillary mucin (PSM) glycoproteins in two different solvents: 0.1M NaCl, and 6M GdnHCl. The relaxation spectrum has been successfully resolved into a slow mode corresponding to pure translational diffusion and a fast mode containing information on the relaxation times for intramolecular motion. Analysis of the slow mode permits a light scattering evaluation of the polydispersity of these high molecular weight mucin glycoprotein fractions. Determination of the longest intramolecular relaxation times tau 1 shows that these are much longer for the PSM fractions in 0.1M NaCl compared to 6M GdnHCl. These data are consistent with earlier studies showing that the chain conformation is the same in both solvents, but that in 0.1M NaCl, the PSM glycoprotein undergoes a self-association process that is end-to-end in nature. Since the tau 1 value is intimately related to the viscoelastic behavior of PSM solutions and gels, it is interesting to speculate that the end-to-end association process may be physiologically important.     

Chemoenzymatic synthesis of ultralow and low-molecular weight heparins

Heparin is a naturally occurring glycosaminoglycan isolated from animal tissues and is medically used as an anticoagulant drug. Adulteration attempts of isolated heparin with chondroitin sulfate in the past resulted in great safety concerns. Also, increasing demands on batch-to-batch homogeneity for better evaluation and control of its pharmacodynamic and pharmacokinetic properties kindled the development of synthetic routes for the production of heparin and its derivatives. The discovery of enzymes involved in glycosaminoglycan biosynthesis and their application in chemoenzymatic synthesis makes it feasible to generate low molecular weight heparins (LMWHs) and ultra-low molecular weight heparins (ULMWHs). Understanding the scope and limitations of these enzymes currently used in the production of synthetic heparins will help to achieve more defined heparins with controlled medicative properties. Here, we summarized the recent advances in the chemoenzymatic synthesis of LMW/ULMW heparins.     

Combination of affinity chromatography and analytical polyacrylamide gel electrophoresis for rapid measurement of human serum high-density lipoprotein apoproteins

Heparin of an average molecular weight of 13,000 was fractionated on the basis of size into five fractions of different weight-average molecular weight ranging from 8500 to 20,000. The heparin was also degraded using microbial heparinase resulting in products ranging from a disaccharide of molecular weight 500 to an oligosaccharide of molecular weight 3100. These products were also size fractionated. The individual heparin fractions and products were tested for metachromatic activity with Azure A. The metachromatic activity of the heparin fractions was independent of molecular weight, while the metachromatic activity of the products was dependent on molecular weight. Metachromatic activity was found in a fragment as small as a tetrasaccharide. Anticoagulant activity was found in fragments of tetrasaccharide or larger by a Factor Xa clotting assay and in fragments of hexasaccharide or larger by a Factor Xa amidolytic chromogenic assay.     

An improved heparin assay which is insensitive to platelet factor 4

A new reagent for the determination of heparin in plasma has been developed. In the assay heparin which was bound to platelet factor 4 is also measured. That is why samples, which have to be assayed for heparin with this reagent, do not need any special pretreatment like fast and cooled processing in order to prevent release of platelet factor 4 from platelets. Heparin can be assayed in samples anticoagulated with citrate which are used routinely for the determination of other coagulation parameters like PT or aPTT. Freezing prior to the assay is possible and does not influence the result. The assay is based on the inactivation of factor Xa by antithrombin III which is catalysed by heparin or smaller fragments of it. It can therefore be used for the determination of heparins of low molecular weight, too. The sample is first mixed with AT III in order to compensate for a potentially decreased level in the probe. Then the F Xa reagent is added, which releases bound heparin from plasma proteins like platelet factor 4 by an added polysulfated dextran simultaneously to the onset of the inhibitory reaction towards F Xa. Free and secondarily released heparin are then available for determination. After a defined period of time a substrate for F Xa is added and the remaining activity is measured in a photometer. An incubation time of 1 min or 3 min is used for the normal range of 0.1 to 1 U/ml or the low dose range from 0.01 to 0.3 U/ml heparin, respectively.     

Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.     

Effect of molecular mass on antitumor activity of heteropolysaccharide from Poria cocos

A water-soluble heteropolysaccharide ac-PCM0 from Poria cocos was successfully fractionated using a preparative size exclusion chromatography (SEC) column, and their weight-average molecular mass (M(w)) was characterized by analytical SEC combined with laser light scattering (SEC-LLS). The results indicate that the fractions having relatively high M(w) exhibited higher inhibition ratio in vivo antitumor activity than those having M(w) below 3.29 x 10(4). However, the relatively low molecular mass was beneficial to the in vitro antitumor activity. Moreover, ac-PCM0 has a significantly higher enhancement ratio of the body weight than 5-fluorouracil, and its 50% lethal dose is above 1250 mg/kg, indicating a nontoxic nature.     

Kinetics of radiolabelled (99mTc) heparin and low molecular weight heparin fractions CY 216, CY 222 in patients with uncomplicated myocardial infarction

Kinetics of plasmatic disappearance of radiolabelled (99mTc) heparin (Choay, Mr mean 15,000) and low molecular weight heparin (LMWH) fraction CY 216 (Choay, Mr mean 5,000) and CY 222 (Choay, Mr mean 4,000) was compared in 2 women and 8 men (aged 50-71, mean 65 years) with uncomplicated myocardial infarction. The three technetiated heparins were consecutively injected intravenously (67 nanomoles) to each of 10 patients, at intervals of 3-5 days, 14-28 days after acute cardiac onset. The plasma radioactivity was counted in blood samples collected within a period of 5 h. Radiolabelled heparin and LMWH fractions CY 216, CY 222 disappeared from plasma following a biexponential clearance curve with a fast and slow component reflecting the biodistribution (alpha) and elimination (beta) phase. The bioavailability values (AUC, t0.5 alpha, t0.5 beta) as well as distribution and elimination rates were similar for all three technetiated heparins. The bulk of injected 99mTc-heparin or LMWH fraction was rapidly distributed to the tissular compartment (t0.5 alpha = 13 min), whereas the radiocomplex remaining in the circulation was slowly eliminated with a half-time (t0.5 beta) of an average 320 min. Radioactivity eliminated from plasma was only partially (30-50%) excreted in the urine. The results indicate that after a low-dose intravenous administration LMWH fractions CY 216 and CY 222 maintain the pharmacokinetics properties of standard heparin, especially the rapid distribution to the tissular compartment.