Anticoagulant and lipolytic effects of a low molecular weight heparin fraction

The lipolytic and anticoagulant actions of a 4000 dalton low molecular weight (LMW) heparin were compared with unfractionated mucosal heparin after intravenous and various subcutaneous doses in man. I.v. injection of 100 USP units/kg body weight lipoprotein lipase (LPL) activity, and inhibition of factor Xa decreased with a half life twice as long after LMW heparin compared to normal heparin (p < 0.05). There were no differences in half lives for HTGL activity, thrombin inhibition and on aPTT. The area under the activity time curve (AUC) of LPL and factor Xa was double with LMW heparin (p < 0.05). S.c. administration showed that the AUC of LMW heparin on the factor Xa inhibition was 10 times larger compared to normal heparin. LPL activity was released comparable to normal heparin. The effects on HTGL were three times larger compared to normal heparin. There were no differences in half lives. The data show that in contrast to normal heparin LMW heparin is rapidly and completely absorbed from the subcutaneous depots. The pharmacodynamic data of LPL activity and factor Xa inhibition suggest similar release mechanisms.     

In vivo release of anti-Xa clotting activity by a heparin analogue

The parenteral injection of a semi-synthetic heparin analogue (SSHA) releases anti-Xa clotting activity, lipoprotein lipase activity and PF₄ antigen. The increased anti-Xa activity is not neutralized by PF₄ or protamine sulphate. A second injection of the drug after 90 minutes, or an increase in dose, does not increase the level of induced anti-Xa clotting activity. Possible mechanisms of action include the release by SSHA of endogenous glycosaminoglycans with anti-Xa activity, and interference by released lipoprotein lipase of a modulator of anti-Xa activity. It is concluded that a drug with weak anticoagulant activity in vitro may nevertheless have significant antithrombotic potential.     

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

Thrombin inactivation on surfaces with covalently bonded heparin

About 8000 Daltons porcine mucosa heparin fragments were covalently bonded by end-point attachment to polyethylene. The interaction between the immobilized heparin, added thrombin, and antithrombin III [AT] was investigated.

The heparin surface was adsorbed with either albumin, AT dissolved in albumin or Tyrode, or platelet free plasma. Irrespective of the pre-treatment procedure, exposure of the surface to thrombin resulted in the same substantial decrease of thrombin in solution and the same degree of surface-confined thrombin activity. It was concluded that the heparin surface has a large capacity to bind thrombin and that the thrombin inhibitory capacity of high affinity heparin fragments is limited.

On exposure of the thrombin-loaded surfaces to defibrinogenated plasma or AT, the surface-confined thrombin was inhibited within 30 seconds. Successive dilutions of plasma or AT decreased the inhibition rate but not the inhibition capacity. It is concluded that inhibition of thrombin adsorbed on the heparin surface occurs as follows: Added AT adheres to high affinity heparin fragments on the surface whereupon adsorbed thrombin migrates in the hydrophilic heparin coating towards the reaction site of AT and becomes inhibited. The inactivated thrombin-AT complex leaves then the surface, thus enabling the process to be repeated.     

Heparin with low affinity to antithrombin III inhibits the activation of prothrombin in normal plasma

Standard unfractionated heparin is known to have two actions on blood clotting. Unfractionated heparin enhances the rates at which antithrombin III inactivates activated clotting factors, and inhibits the activation of both Factor X and prothrombin by disrupting the calcium and phospholipid dependent assembly of the Factor X and prothrombin activator complexes. This latter inhibitory action of heparin occurs independently of antithrombin III. A heparin fraction with low affinity to antithrombin III was prepared from standard heparin by affinity chromatography on antithrombin-III-Sepharose and its properties compared with unfractionated heparin. The low affinity heparin fraction and the unfractionated heparin had equivalent inhibitory effects on prothrombin activation in antithrombin III depleted plasma. In normal plasma, the low affinity fraction inhibited the activation of prothrombin. Unlike the unfractionated heparin, however, the fraction of heparin with low affinity to antithrombin III did not enhance the inactivation of either Factor Xa or thrombin. This antithrombin III independent inhibition of the activation of prothrombin was also evident when activated platelets were used as the source of the procoagulant phospholipids. The antithrombin III independent effect of heparin is unlikely to be important therapeutically, however, if this property of heparin is shared by other naturally occurring glycosaminoglycans, it could be important in maintaining the fluidity of blood under physiological conditions.     

The effects of antithrombin III, heparin and a novel heparinoid infusions on the bleeding tendency using an experimental model in the rat

The effects of selective increase of plasma AT-III concentration (to 2 U/ml and 8 U/ml) in the presence or absence of low dose commercial heparin and a novel natural heparinoid (Org 10172) on APTT, clotting factors and bleeding were investigated in an experimental model in rats.

Slight increases in the APTT were observed with: a. Org 10172, b. both AT-III doses and c. combinations hereoff. However, synergism was observed in the prolongations of the APTT when the AT-III concentrate (70 U/kg and 500 U/kg) was combined with both heparin dosages. AT-III concentrates (70 U/kg and 500 U/kg) or Org 10172 (2,5 mg/kg) separately or in combination showed no effect on bleeding. Heparin (0,125 and 0,25 mg/kg i.v.) also showed no effect on bleeding neither if they were combined with the lower dose AT-III. However, heparin (both dosages) combined with 500 U/kg of AT-III concentrates significant increased blood loss.

These observations suggest that infusion of AT-III concentrate additionally to low dose heparin therapy did not increase bleeding in the rat model used provided that extreme high AT-III plasma levels (8 U/ml) were avoided. The novel natural heparinoid Org 10172 alone or combined with either AT-III dosage induced no increased bleeding.     

Laboratory monitoring of thromboprophylaxis with low molecular weight and standard heparin

This study was made to evaluate assays for monitoring of low dose heparin thromboprophylaxis and to evaluate its efficacy in reduction of hypercoagulation. Patients with medical diseases scheduled for routine thromboprophylaxis were subcutaneously treated with either 5.000 anti XaU low molecular weight (LMW) heparin once daily (n=20) or 5.000 IU standard (ST) heparin 3 times daily (n= 19). On days 1,2,3, before, 1 and 4 hours after heparin injection APTT, TCT, anti Xa, Heptest, thrombin-antithrombin complexes (TAT), and D-Dimer levels were measured. In the LMW heparin group, median values of APTT and TCT slightly increased after heparin and the ranges of pre- and postinjection values showed extensive overlap. However, values of anti Xa and Heptest markedly increased, showing complete separation of ranges. In the ST heparin group neither APTT, TCT, anti Xa, nor Heptest were significantly different comparing pre- and postheparin values. Half of the patients in both groups had subclinical hypercoagulation at baseline (TAT>5ng/ml, D-Dimer>200ng/ml). On day 3 of prophylaxis this percentage was not significantly decreased. Moreover, several patients in both groups increased in TAT and D-Dimer. In the LMWheparin group, negative correlations between body weight and 4 h postinjection heparin levels were found (anti Xa R=−0.50, Heptest R=−0.31) and between 1 h postinjection heparin and TAT and D-Dimer levels 3 h later (TAT-anti Xa R=−0.58, TAT-Heptest R=−0.64, D-Dimer-anti Xa R=−0.32, D-Dimer-Heptest R=−0.33). These results show that low dose LMW but not ST heparin therapy can be monitored by the anti Xa test or the Heptest.     

Pharmacokinetics of a low molecular weight heparin, Logiparin, after intravenous and subcutaneous administration to healthy volunteers

In six healthy volunteers we have estimated the pharmacokinetic parameters of the anti factor Xa (AXa) and anti factor IIa (AIIa) activities of a LMW heparin, Logiparin. For the AXa the following parameters were estimated in a 1-compartment model (mean and 95% confidence limits in brackets): elimination half life 82 minutes (60–127 min), absorption half life (s.c.inj.) 200 minutes (137–368 min), bioavailability 90% (24–156 %), and apparent volume of distribution 3.9 1 (3.1–5.2 1). The plasma activity was linearly correlated to the dose given and to the body weight of the volunteer. For the AIIa the parameters estimated in a 1-compartment model were: elimination half life 71 minutes (52–115 min), absorption half life 257 minutes (133–3442 min), bioavailability 67% (44–90 %), and apparent volume of distribution 10.1 1 (7.2–16.7 1). The plasma activity was dependent on dose and body weight but it also seemed to be influenced by individual factors. This study shows that the absorption rate is the rate limiting factor and the explanation for the long lasting effect of this LMW heparin after subcutaneous injection. The slow absorption rate and the high bioavailability are probably the major advantages of LMW heparins compared to conventional heparin.     

Studies on a highly active anticoagulant fraction of high molecular weight isolated from porcine sodium heparin

We have studied heparin fractionation using gel filtration and ion-exchange chromatographic methods. The starting material was commercial grade porcine mucosal sodium heparin (PSH). The fractionation was monitored employing synthetic substrates for assaying both antithrombin (with H-D-Phe-Pip-Arg-pNA ; S-2238) and anti-FXa (with Bz-Ileu-Glu-Gly-Arg-pNA ; S-2222) activities. The resulting fractions were evaluated in different amidolytic and coagulation methods used to determine heparin potency by comparison with PSH. By gel filtration of PSH on Ultrogel AcA 54, both strong anti-FXa and antithrombin activities were associated with the fractions eluted in the high molecular weight range (MW 20 × 10³). These fractions also had potent anticoagulant action when assayed by conventional clotting methods. PSH was also subjected to fractionation by an ion-exchange technique (DEAE-Sephacel) with increasing salt molarity. The patterns for antithrombin and anti-FXa activities were again closely related, if not identical. Four fractions were usually distinguished, with respectively negligible, intermediate, high and very high activities when compared to PSH. The very highly active fraction (HAF), approximately 15% by weight, was eluted at high salt molarity (> 0.8 M NaCl). On a weight basis its anticoagulant activity was 2–3 times that of PSH as determined by amidolytic as well as clotting methods. Intravenous injection of HAF to rabbits and dogs (1.0 and 2.5 mg/kg) produced a much stronger anticoagulant response than PSH, also showing an effect which persisted for a longer duration.     

Importance of a 3-0-sulfate group in a heparin pentasaccharide for antithrombotic activity

Previous theoretical and experimental evidence led to the formulation of a specific pentasaccharide structure which represents the site in heparin for binding to antithrombin III. This pentasaccharide was subsequently synthesized. A pentasaccharide of the same structure but lacking only the sulfate group on the hydroxyl group of the middle glucosamine (position C-3) was also synthesized to test the structure - activity relationships. Previous biochemical studies showed the 3-0-desulfated pentasaccharide to have a low affinity binding to AT III and to be devoid of the high anti-factor Xa activity characteristic of the pentasaccharide. Our in vivo studies, in a venous stasis thrombosis model proved the 3-0-desulfated pentasaccharide, at equigravimetric dosages, to be devoid of the antithrombotic activity previously reported for the pentasaccharide. These studies confirm the fact that inhibition of factor Xa at a high level of activity produces an antithrombotic effect.     

Heparin-induced platelet aggregation: Dose/response relationships for a low molecular weight heparin derivative (pk 10169) and its subfractions

Addition of heparin or heparin derivatives to citrate anticoagulated platelet-rich plasma caused platelet aggregation in a dose-dependent manner. Utilizing heparin, a low molecular weight heparin derivative (PK 10169) and its various subfractions, we determined dose/response relationships for platelet aggregation and found that the ability of these agents to cause platelet aggregation was dependent upon the molecular weight of the individual subfraction used. In comparison to unmodified porcine mucosal heparin, the lower molecular weight derivative (PK 10169) yielded a dose/response curve that was shifted down and to the right, and indicated that this agent was less potent in causing platelet aggregation. In addition, as the molecular weight of PK 10169 subfractions decreased, their dose/response curves were progressively shifted down and to the right. The lowest molecular weight subfraction was essentially without platelet aggregating activity. We also measured the anti IIa and anti Xa activities of these agents and concluded that these activities did not appear to correlate with platelet aggregating activity. Platelet aggregation studies with PK 10169 subfractions of high and low affinity for antithrombin III (AT III) indicated that the platelet aggregating activity of these compounds may not be related to their affinity for AT III, but results were not definitive.     

Antithrombin activity of a fucan sulfate from the brown seaweed Ecklonia kurome

The mechanism of antithrombin action of a fucan sulfate (C-II), which was isolated from the brown seaweed Ecklonia kurome, was examined by clotting method using a thrombin-fibrinogen system and by amidolytic method using a chromogenic substrate in the presence and the absence of antithrombin III (AT III) or heparin cofactor II (HG II). C-II significantly inhibited the clotting of fibrinogen by thrombin even in the absence of the protease inhibitors, and the anidolytic activity of the protein only in the presence of HC III. C-III was not adsorbed on an AT III-agarose column and its anticoagulant activity in AT III-depleted plasma was the same as that in normal one. Examination of interaction of C-III with fibrinogen by g-el filtration chromatography demonstrated that C-III bound to the protein. These results indicated that the antithrombin activity of C-III was mediated by HC III and not by AT III, and that the polysaceharide bound to fibrinogen, thereby blocking thrombin action, and also that its direct thrombin inhibition was very weak.     

Comparative study of antithrombotic effect of a low molecular weight heparin and unfractionated heparin in an ex vivo model of deep arterial injury

Thrombosis after plaque rupture triggers the onset of acute coronary events. The treatment of choice for patients with acute coronary syndromes is conventional unfractionated heparin. Low molecular weight heparin has recently been reported to be as effective and even safer than unfractionated heparin. In this study, the effects of the low molecular weight heparin reviparin and unfractionated heparin on thrombus formation were examined under dynamic conditions using an extracorporeal perfusion chamber in a porcine model. Thrombus formation was assessed by the deposition of porcine ¹²³I-fibrin(ogen) and autologous ¹¹¹In-platelets on porcine tunica media at high and low shear rates. Reviparin reduced the fibrinogen molecules deposited on injured vessels at high shear rates (252±80 molecules×10¹²/cm² for reviparine (200 U/kg/hour) vs. 624±70×10¹²/cm² for unfractionated heparin (200 U/kg/hour) (p<0.05). At low shear rates, fibrinogen deposition was also significantly reduced by reviparin (130±15 molecules×10¹²/cm²) compared to unfractionated heparin (192±40×10¹²/cm² at 200 U/kg/hour; p<0.05). No change in platelet deposition was detected after heparin administration in either treatment group. In conclusion, the low molecular weight heparin reviparin has a higher antithrombotic potential than unfractionated heparin. Reviparin may have advantages over unfractionated heparin in treatment and prevention of acute coronary syndromes.     

Comparative effects of heparin and PK 10169, a low molecular weight fraction, in a canine model of arterial thrombosis

The comparative properties of heparin and PK 10169, a low molecular weight fraction, were studied using an antithrombotic test in anaesthetized dogs. The antithrombotic properties of the two compounds were evaluated by measuring inhibition of thrombus formation following transluminar stimulation of coronary artery with anodal current and by measuring anticoagulant properties, anti Xa and anti IIa activities. The results show that PK 10169 displayed significant antithrombotic activities above 0.625 mg/kg and was equipotent at 2.5 mg/kg s.c. with heparin 10 mg/kg s.c. No correlation could be observed between antithrombotic/anti Xa ratio of both compounds. Moreover it was shown that, unlike heparin, PK 10169 s.c. was devoid of obvious anticoagulant properties and induced a negligible anti IIa activity contrasting with a high anti Xa level. A similar dissociation between anti Xa and anti IIa activities was observed following i.v. administration of 2.5 mg/kg of PK 10169 but not with heparin. This low molecular weight heparin fraction might thus be regarded as a potential arterial antithrombotic agent devoid of appreciable anticoagulant effect.     

Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: Evaluation by an amidolytic assay

The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium. Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium. We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E. coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA). Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10⁶ neutrophils per ml), had major effects on endothelial SAA. When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% ± sem 7.8; p< 0.02) or purified ATIII (mean percentage of control 69% ± sem 4.6; p< 0.001). We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function. This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.     

Lack of correlation between "in vitro" and "in vivo" antithrombotic activity of heparin fractions and related compounds. Heparan sulfate as an antithrombotic agent "in vivo"

The anticoagulant (U.S.P., APTT; "in vitro" and "in vivo") antithrombotic (aXa; Yin and Wessler and chromogenic), antilipemic (LPL) activities of heparin, heparin fractions and fragments, heparinoids, heparan sulfate and other sulfated glycosaminoglycans were compared with the activities of these compounds as antithrombotics "in vivo" by four different methods (vena cavae ligature, kaolin, collagen and steel coil). A lack of correlation was observed between the activities "in vitro" and the antithrombotic activity "in vivo". For instance heparan sulfate which shows negligible pharmacological activities "in vitro" is a potent antithrombotic agent "in vivo". Likewise, several heparin fractions and fragments have low aXa activity "in vitro" and high antithrombotic activity "in vivo". It is concluded from these results that the "in vitro" tests used cannot predict the antithrombotic activity "in vivo".     

Isolation and anticoagulant properties of a new sulfated xylan: Comparison with heparin and a sodium pentosan polysulfate (SP-54)

Larchwood xylan was purified by diaminoethylaminoethyl (DEAE) cellulose chromatography, sulfated by heating with chlorosulfonic acid -pyridine complex and the sulfated polysaccharide was isolated by epichlorohydrin triethanolamine (ECTEOLA) cellulose chromatography as the sodium salt. It's molecular weight was 25000 and the specific rotation was []_D20–70°. Electrophoretic analysis of the sulfated xylan along with heparin and SP-54 using lithium acetate-agarose technique showed that the charge density of the xylan sulfate was similar to heparin and it moved as a single component in contrast to commercial heparin which resolved into two while SP-54 moved at the same rate as the marker dye. Anticoagulant properties of the sulfated xylan were compared with heparin and SP-54 by studying its effect on the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) using pooled normal human plasma. The sulfated xylan was more effective than SP-54 in delaying coagulation by all the three measurements while it was less active than heparin in inhibiting APTT and PT.