Anticoagulant activities of pentosan polysulphate (Hémoclar) due to release of hepatic triglyceride lipase (HTGL)

Subcutaneous injections of 50 mg pentosan polysulphate (Hémoclar) were given to normal volunteers and the effects on anti-Factor Xa activity, thrombin generation and lipase release measured. Concentrations of pentosan polysulphate were measured by a competitive binding assay and the mean peak level found to be 1.6 micrograms/ml. Anti-Xa clotting activity rose to 0.034 iu/ml and thrombin generation induced by lipid peroxides was inhibited by approximately 50%. Neither of these effects could be accounted for by the direct action of pentosan polysulphate at the concentrations measured. Pentosan polysulphate was very effective in releasing lipase, approximately 70-80% of the total enzyme activity being due to hepatic triglyceride lipase (HTGL). In vitro addition of purified HTGL to plasma markedly enhanced anti-Xa clotting activity, and caused a 70% inhibition of lipid peroxide induced thrombin generation. Anti-Xa activity of post-injection plasma was increased rather than neutralised by addition of polybrene, and this effect could be mimicked by addition of polybrene to plasma containing pentosan polysulphate and purified HTGL. It is concluded that, when given in low doses subcutaneously, pentosan polysulphate acts as an indirect anticoagulant, its major effects being due to release of HTGL.     

Release of lipoprotein lipase and hepatic triglyceride lipase in rats by heparin and other sulphated polysaccharides

The ability of parenteral heparin to release the capillary-bound enzymes lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) into the circulation is shared by several other sulphated polysaccharides. The lipase-releasing activity in rats of two unfractionated heparins has been compared with that of characterised preparations of other glycosaminoglycans and of two partially synthetic polysaccharides. The results suggest that whilst both molecular weight and degree of sulphation are important in determining the potency, there is also some specific structural element associated with the heparin class. The clearance of lipases was also investigated. The half-lives of LPL (29 mins) and of HTGL (36 mins) did not appear to be affected by the nature of the releasing agent.     

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 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.     

Inhibition of low molecular weight heparin by protamine chloride in vivo

To determine the antagonization of anticoagulant and lipolytic effects of a low molecular weight [LMW] heparin preparation protamine chloride was given intravenously after i.v. injection of LMW or normal heparin. The effects of normal heparin on factor Xa, thrombin, aPTT, lipoprotein [LPL] and hepatic triglyceride lipase [HTGL] activities were neutralized immediately by i.v. protamine. The inhibition of thrombin and aPTT by LMW heparin were also abolished, whereas the effects on LPL and HTGL were counteracted to 80% and on factor Xa only to 40% by i.v. protamine chloride. No rebound of the anticoagulant or lipolytic effect was detected. It is assumed that haemorrhagic complication during therapy can be antagonized by protamine chloride. The incomplete inhibitory effect of protamine chloride on LPL, HTGL and factor Xa activities of LMW heparin indicate that protamine chloride requires more than 14 saccharide units in the heparin molecule for interaction.     

Neutralisation of heparan sulphate and low molecular weight heparin by protamine

The neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.     

Studies of anti-Xa activity in human plasma II: The role of lipoproteins

The major plasma inhibitor of Factor Xa is thought to be anti-thrombin III (At III). However, adsorption of plasma by aluminium hydroxide (A1(OH)₃) increases its rate of neutralisation 7–8 fold, and this ‘fast-acting’ anti-Xa activity has been shown to be independent of At III. Gel filtration of plasma indicated that the anti-Xa activity after Al(0H)₃ adsorption was located largely in the high molecular weight (> 200,000) fractions, which contain most of the plasma lipoproteins. Purified lipoproteins of very low-density (VLDL), low-density (LDL) and high density (HDL) were prepared by ultracentrifugation and their anti-Xa activities measured before and after adsorption by Al(OH)₃. Both LDL and HDL had significant anti-Xa activities by clotting and amidolytic assays. Al(OH)₃ adsorption of LDL and HDL gave a marked increase in anti-Xa clotting activity and a decrease in amidolytic activity. Incubation of the adsorbed lipoproteins with phospholipase enzymes destroyed the anti-Xa activity, and prior incubation of Factor Xa with Ca⁺⁺ and phospholipid protected it against inactivation, indicating that the anti-Xa activity of the adsorbed lipoproteins is mediated via binding of Xa to phospholipid in the lipoproteins. These results indicate that lipoproteins, especially LDL and HDL, are responsible for the increased anti-Xa activity of plasma after Al(OH)₃ adsorption. These lipoproteins appear to contain high affinity phospholipid binding sites for Xa which are revealed by Al(OH)₃ adsorption.     

Comparative study of the pharmacokinetic profiles of two LMWHs--bemiparin (3500 IU,anti-Xa) and tinzaparin (4500 IU,anti-Xa)--administered subcutaneously to healthy male volunteers

Pharmacokinetic profiles of bemiparin (3500 IU, anti-Xa) and tinzaparin (4500 IU, anti-Xa) administered subcutaneously to 12 healthy male volunteers were compared in a monocentric study. Each of the 12 subjects underwent successively the two low-molecular-weight heparin (LMWH) preparations in a randomised order and was considered as its own control. Anti-Xa activity, free and total tissue factor pathway inhibitor (TFPI), and thromboplastin-thrombomodulin-mediated time were determined as main variables. Activated partial thromboplastin time (APTT), thrombin clotting time, and anti-IIa activity were also determined. Bemiparin (3500 IU, anti-Xa) exerts a significantly more rapid, more potent, and more prolonged anti-Xa activity than tinzaparin (4500 IU, anti-Xa). The plasma level increase for free and total TFPI is significantly lower with bemiparin than with tinzaparin. Free and total TFPI peak levels occur earlier than anti-Xa activity peak levels for both LMWH preparations, but no statistical difference appeared between the two preparations for TFPI T(max). No significant effect was observed for both preparations for thromboplastin-thrombomodulin-mediated time. Subcutaneous injection of bemiparin exerts only minimal anti-IIa activity and does not prolong thrombin time, whereas tinzaparin elicits significant anti-IIa activity and prolongs thrombin clotting time. Bemiparin exerts a significantly lower prolongation of APTT than tinzaparin. No difference was observed for APTT prolongation T(max) between the two preparations. Globally, the overall tolerability of both formulations revealed no relevant adverse effects. In conclusion, bemiparin and tinzaparin are not bioequivalent. Bemiparin exerts an important and more prolonged anti-Xa activity in comparison with tinzaparin. An original finding of this study is the difference observed between the two formulations for free TFPI release.     

Use of the pentasaccharide fondaparinux as an anticoagulant during haemodialysis

No data about the use of the pentasaccharide fondaparinux, a highly selective indirect inhibitor of factor Xa, in patients treated with haemodialysis are available. Therefore, we investigated the pharmacokinetics and -dynamics of fondaparinux in 12 patients during haemodialysis. The anti-Xa activity (expressed as fondaparinux equivalent) was monitored, a semiquantitative clotting scale (SQCS) ranging from 0 (no visible traces of coagula) to 3 (complete clotting of the dialysis circuit) was applied, and the digital compression time necessary to achieve haemostasis at the puncture site was determined. After an initial period, when the regular heparin dose was replaced once weekly by fondaparinux, 0.05 mg/kg, the pentasaccharide was administered for nine consecutive haemodialysis sessions. Peak anti-Xa activity increased from 0.61 +/- 0.14 microg/l after the first dose to 0.89 +/- 0.24 microg/l after dose 9 (P < 0.001), whereas predialysis anti-Xa activity steadily rose to 0.32 +/- 0.09 microg/l (P < 0.001). A sufficient but slightly less effective anticoagulation with a mean SQCS of 1.19 +/- 0.71 (n = 121) was obtained by fondaparinux as compared with 0.65 +/- 0.58 (n = 60, P < 0.005) by 4,825 +/- 1,703 U of unfractionated heparin. Mean digital compression time rose slightly during fondaparinux from 23.7 +/- 7.4 minutes to 24.8 +/- 7.5 minutes (P < 0.05) and, more important, six of the 12 patients reported minor bleeding problems during the interdialytic interval. Thus, fondaparinux can be used to prevent circuit clotting during haemodialysis; however, accumulation results in an interdialytic increase of anti-Xa activity. Therefore, fondaparinux should be reserved for patients requiring systemic anticoagulation on the days off dialysis.     

In vitro effect on Heptest of low molecular weight heparin fractions and preparations with various anti-IIa and anti-Xa activities

The Heptest heparin assay has recently been introduced, and evaluated for the laboratory monitoring of patients receiving low molecular weight heparins (LMWH). The aim of the present study was to elucidate the relative role on the Heptest assay of the anti-factors Xa and IIa activities present in the three types of compounds that possess: 1. exclusively anti-Xa activity (LF1: LMWH fractions with MW ranging from 1,200 to 4,200 D.); 2. both anti-Xa and anti-IIa activities (LF2 with MW from 4,800 to 12,000 D.); 3. exclusive anti-IIa activity (Hirudin and Dermatan Sulfate). All compounds studied demonstrated dose-dependent activities in both amidolytic and clotting assays. The LF2 in contrast to the LF1, additionally enhanced the clotting times of Heptest. This enhancement was shown to be due to the anti-Factor IIa activity of the agents. Heptest does not exclusively reflect Anti-Xa activity since it is influenced by agents containing exclusive anti-IIa activity like Hirudin and Dermatan Sulfate. At low concentrations of LF2, Heptest measures predominantly the anti-factor Xa activity while at higher concentrations it is influenced by the combined activity of anti-factor Xa and anti-factor IIa. However, Heptest sensitivity to anti-factor IIa is significantly lower than for anti-Xa activity.     

Anticoagulant properties in vitro of heparan sulphates

The anticoagulant properties in vitro of eight heparan sulphate preparations were studied using clotting (APTT, anti-Xa) and amidolytic (anti-Xa, anti-thrombin) assays. Activities ranged from very low levels (less than 5 iu/mg) up to values similar to those of heparin. Activities measured by APTT assay showed the best correlation with the sulphate to carboxylate ratio of the heparan sulphates. Highest activities were obtained in the anti-Xa clotting assay, these being approximately two-fold greater than activities in the anti-Xa amidolytic assay. Five of the heparan sulphate preparations were readily neutralised by protamine sulphate, whereas the three heparans with the lowest sulphate to carboxylate ratio were much more resistant to neutralisation. After fractionating each heparan sulphate into At III-binding and non-binding material, it was found that the anti-coagulant properties were associated only with the former. It is concluded that these properties are dependent on the activation of At III.     

Triglyceride lipase activity and human platelets.

The activity of triglyceride lipases in human postheparin plasma is significantly higher in platelet rich than platelet poor plasma. This holds for total activity, lipoprotein lipase (LPL) activity, and hepatic triglyceride lipase (H-TGL) activity. Gel filtration of platelet rich postheparin plasma on Sepharose 2 B will separate platelets from triglyceride lipase activity. The very small triglyceride lipase activity of isolated platelets is inhibited by 1.0 M NaCl, slightly inhibited by specific antibody to hepatic lipase, and not influenced by specific antibody to lipoprotein lipase.     

Anticoagulant activities of four unfractionated and fractionated heparins

Four commercial heparins, two of bovine lung and two of porcine mucosal origin, have been fractionated by gel filtration (and one of them also by ion-exchange chromatography) to yield different fractions whose mean MW_s lie in the MW range 7,000–26,000. Three plasma anticoagulant assays have been used to measure the specific activities of the heparin fractions, a kaolin-cephalin clotting time method (KCCT), a calcium-thrombin clotting time method (TCT) and a specific anti-factor Xa assay. The two unfractionated mucosal heparins were found to have higher specific activities when measured in the anti-Xa assay compared to the KCCT assay, while the unfractionated beef lung heparins had lower anti-Xa than KCCT activities. An increase with MW of the heparin fractions was found in specific activities as measured by KCCT and TCT assays over most of the MW range. The low MW heparin fractions (less than 10,500) from all of the heparins had specific activity ratios, KCCT:anti-Xa and TCT:anti-Xa, which were always less than unity. These specific activity ratios increased with increasing MW, usually to greater than unity for the highest MW (greater than 22,000) fractions. There was a tendency for these specific activity ratios to be higher for the beef lung than the mucosal heparin fractions at a given MW. The ability of heparin fractions to inhibit the thrombin induced aggregation of platelets in citrated platelet-rich plasma increased with MW over most of the MW range studied and seemed to follow the specific activities of the fractions when measured by TCT or KCCT assays.     

Monitoring high-dose heparinization during cardiopulmonary by-pass--a comparison between prothrombinase-induced clotting time (PiCT) and two chromogenic anti-factor Xa activity assays

Heparinization requires monitoring, but optimal methods for measuring the anticoagulant effects of heparin remain to be determined. We compared prothrombinase-induced clotting time (PiCT) and two chromogenic anti-factor Xa activity (anti-Xa) assays in monitoring high-dose heparinization during cardiopulmonary by-pass (CPB). Heparin effects were serially measured with PiCT and two anti-Xa assays in 100 patients. Antithrombin and protein C activities were measured preoperatively, and antithrombin activity was measured during CPB. Activation of coagulation was assessed with measurements of prothrombin fragment F1+2, soluble fibrin complexes, and D-dimer before, during, and after CPB. During CPB mean ranges of PiCT and of anti-Xa heparin levels measured with (anti-Xa A) and without (anti-Xa B) dextran sulfate and antithrombin supplementation were 5.0-5.2, 4.7-5.0, and 4.5-4.9 IU/ml, respectively. There was poor agreement between PiCT and anti-Xa and between the two anti-Xa assays (r = 0.32-0.65 and broad limits of agreement). Patients with low preoperative antithrombin or protein C levels had lower PiCT (p = 0.028 and p = 0.01) and anti-Xa A (both p<0.001) levels during CPB than others. Patients with the lowest heparin activities during CPB (lowest deciles of PiCT and anti-Xa A) had higher subsequent F1+2 after CPB (p = 0.002 and p = 0.02), and patients with high heparin levels required fewer transfusions of packed red blood cells than others. In conclusion, in the challenging setting of CPB there is poor agreement between anti-Xa assays and PiCT. However, coagulation-based PiCT could provide an alternative to the chromogenic anti-Xa assays. Higher heparin levels during CPB were confirmed to associate with reduced transfusion requirements.     

Absorption of heparin, LMW heparin and SP54 after subcutaneous injection, assessed by competitive binding assay

Unfractionated heparin, pentosan polysulphate (SP54) and the low molecular weight heparins CY216 and CY222 were injected subcutaneously at a minimum of weekly intervals into 5 healthy volunteers. The dose was 75 mg in all cases. Concentrations of administered glycosaminoglycan in serial plasma samples and voidings of urine were measured using a competitive binding assay, and biological activity was assessed in plasma using APTT and anti-Xa clotting assays. There was wide individual variation in the absorption of unfractionated heparin as indicated both by the maximal plasma concentrations reached 2-3 h after injection and by the area under the concentration vs. time curve. The efficiency of absorption increased and the individual variation decreased with decreasing molecular weight of the administered glycosaminoglycan. Urinary excretion correlated with plasma concentration, and recovery in the urine also increased with decreasing molecular weight. Similar patterns of uptake and clearance were indicated by the APTT and competitive binding assays, but anti-Xa clotting activity could be detected in the plasma after clearance of the administered glycosaminoglycan.     

Plasma lipolytic activity after subcutaneous administration of heparin and a low molecular weight heparin fragment

The effect of heparin and a low molecular weight heparin fragment (LMWH, mean molecular weight 4000-6000) on plasma anticoagulation and lipolysis was studied in eight healthy men. The activities of antifactor Xa (antiFXa), lipoprotein lipase (LPL), hepatic lipase (HL) and plasma levels of free fatty acids (FFA) were analysed after the injection of 5000 antiFXa units of heparin or LMWH subcutaneously. In comparison with heparin, the administration of LMWH resulted in a significantly higher antiFXa activity (p less than 0.001) but a lower release of LPL and HL (p less than 0.001), which did not increase plasma FFA. It is concluded that subcutaneous injection of LMWH in men elicits an adequate anticoagulant effect measured as antiFXa activity but has a negligible effect on plasma lipolytic activity.     

Molecular weight dependence of the anticoagulant properties of heparin: Intravenous and subcutaneous administration of fractionated heparins to man

A commercial mucosal heparin preparation has been fractionated by gel filtration into five different MW fractions of approximate mean MWs 11500–21500. These five heparins were randomly administered to 5 normal volunteers by intravenous and subcutaneous injection. The anticoagulant effects of the fractions were then determined by KCCT, anti-Xa clotting and anti-Xa chromogenic substrate assays. Following intravenous injection the high MW fraction produced identical elimination curves in all three assays, but with decreasing MW there was a divergence between results obtained by KCCT and anti-Xa assays. The lowest MW fraction produced between 2–3 times greater heparin levels when measured by anti-Xa assay. This divergence in assay results was produced as a consequence of the differences in specific activities seen when fractionated heparins were assayed in vitro by the different assay methods. That is, low MW heparin fractions had a higher anti-Xa specific activity than that determined by the KCCT assay. Conversely, high MW heparin had a greater specific activity when determined by the KCCT assay. When the response to intravenous heparin injection (measured by KCCT and anti-Xa assays) was compared to the dose administered it was found that the anti-Xa response was greater than expected. This increased plasma heparin like activity was not dependant upon the MW of the heparin fractions, and could not be neutralised by PF₄. A MW dependance was observed for the absorption of the heparin fractions into the circulation following subcutaneous injection, with low MW heparin producing higher heparin levels determined by all the assays.     

Anticoagulant effects of a low molecular weight heparinoid (Org 10172) in human volunteers and haemodialysis patients

Org 10172, a low MW heparinoid derived from animal intestinal mucosal tissue, has a mean molecular weight of 6500 D and a specific activity of 8.0 anti-Xa U/mg. Its elimination half-life after i.v. administration is 18 hours. Six human volunteers received repeated single i.v. injections of 800 and 3200 anti-Xa units of Org 10172, 5000 IU heparin or placebo. Bleeding time, platelet count and plasma beta thromboglobulin were not affected by Org 10172 or heparin. Heparin stimulated ADP-induced platelet aggregation (0.2 uM; p less than 0.05) and inhibited thrombin induced aggregation (0.3 U/ml; p less than 0.05), while the heparinoid lacked these effects. Heparin increased plasma platelet factor 4, whereas Org 10172 had no effect. In contrast to heparin Org 10172 had only a minor effect on the activated partial thromboplastin time and thrombin time, while both compounds induced anti-Xa activity in plasma. In a crossover study in six haemodialysis patients, both heparin and Org 10172 (34.4 and 22.4 anti-Xa units/kg/body weight) successfully prevented clotting of the extracorporeal circuit. Microscopical analysis of the artificial kidney membranes showed that the 34.4 unit Org 10172 dosage was as effective as heparin in preventing fibrin deposition. The haemostatic and coagulation effects were as expected from those observed in the volunteers except that there was a slower elimination of the plasma anti-Xa response. In addition heparin and Org 10172 (34.4 anti-Xa units/kg) inhibited the Xa-induced platelet aggregation (0.5 U/ml; p less than 0.01 and p less than 0.001 respectively).     

In vivo studies on the inhibition of coagulation by fractionated heparin and by a heparin analogue. II. Effects of a heparin analogue

SSHA, a semi-synthetic heparin analogue, and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six healthy volunteers over a period of three days in a cross-over trial. Before injection and 2, 4, 6, 8 hrs afterwards, the heparin-like activity was measured with the APTT, the anti-Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induced proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). Repeated injections of SSHA twice daily led to increasing effects on two tests for heparin-like activity, whereas two other tests remained unchanged. Both drugs were tolerated equally well, side effects were not detected. Clinical studies are required to demonstrate whether SSHA is similar or superior to low-dose heparin for use in thrombosis prophylaxis.     

The expression and activity of brain lipoprotein lipase is increased after acute cerebral ischemia‐reperfusion in rats

Lipoprotein lipase (LPL) is a key enzyme involved in lipid metabolism. Previous studies have shown that the levels of brain LPL mRNA, protein and activity are up‐regulated after brain and nerve injury. The aim of this study was to determine the response of expression and activity of brain LPL following acute cerebral ischemia‐reperfusion. Adult male Sprague‐Dawley rats were subjected to surgical occlusion of the middle cerebral artery. The expression of brain LPL was assessed by immunohistochemical staining and the enzyme activity of brain LPL was evaluated by colorimetric method. Increase of LPL immunopositive cells in the cerebral cortex around the infarction area was observed at 4, 6, 12 h ischemia, 2 h ischemia 2 h reperfusion, and 4 h ischemia 2 h reperfusion. LPL activity was significantly decreased in the ischemic side cortex at 2 h ischemia, and then significantly increased at 4 and 6 h ischemia. Our results showed that LPL immunopositive cells were increased in the cortex around the infarction area, and activity of LPL first decreased and then increased following acute cerebral ischemia‐reperfusion. These results may suggest that LPL plays a potential role in the pathophysiological response of the brain to cerebral ischemia‐reperfusion.