Pharmacokinetics of heparin and low molecular weight heparin fragment (Fragmin) in rabbits with impaired renal or metabolic clearance

The kinetics and tissue distribution of 3H-heparin and a 3H-labelled low molecular weight heparin fragment were compared in normal rabbits as well as in rabbits with blocked renal function or reticuloendothelial system (RES). Radioactivity in plasma, urine, liver and kidneys, as well as anti-FXa activity in plasma were determined. The plasma elimination of heparin was, when compared to normal controls, prolonged both in rabbits with renal dysfunction as well as in rabbits with blocked RES, while renal dysfunction was the only parameter that significantly prolonged the plasma half-life of Fragmin. Studies on tissue distribution in normal rabbits revealed that about 60 per cent of the radioactive heparin dose accumulated in the liver and kidney three hours after the injection, whereas the corresponding value was less than 10 per cent for the Fragmin-derived radioactivity. The recovery of radioactivity in urine within three hours was 5 and 35 per cent of the dose, respectively, for 3H-heparin and 3H-Fragmin. It is concluded from the present study that the rapid plasma elimination of heparin in the rabbit (t1/2 = 17 minutes) is mainly due to a high tissue distribution (liver and kidney) while the plasma elimination of Fragmin (t1/2 = 28 minutes) is mainly caused by renal excretion.     

Bleeding times in rats treated with heparin, heparin fragments of high and low anticoagulant activity and chemically modified heparin fragments of low anticoagulant activity

A template bleeding time study in the rat was undertaken to see if it is possible to correlate bleeding times with the molecular weight, anticoagulant activity or chemical composition of heparin or heparin-derived compounds. Heparin from porcine intestinal mucosa (PM-heparin) and from bovine lung (BL-heparin) as well as heparin fragments from these sources were compared. Heparin fragments of low anticoagulant activity were prepared by affinity chromatography on immobilized antithrombin as well as by chemical modification. A heparin fragment of high affinity for antithrombin (HA-fragment) caused a marked and dose-dependent increase in bleeding time while the corresponding heparin fragment with low affinity for antithrombin (LA-fragment) had a marginal and non-dose dependent effect on the bleeding time. Similar results were also obtained with PM-heparin with high and low affinity for antithrombin. A high anti-FXa activity was not always correlated with a marked bleeding tendency. Provided that a fragment was devoid of activated partial thromboplastin time (APTT) activity, it was not possible to provoke a bleeding time of 20 min or longer, although the compound was administered at a dose of 1,088 U/kg (anti-FXa activity). On the other hand, a N-acetylated chemically oversulphated heparin fragment, with a very low anti-FXa activity (1 U/mg) and with an APTT activity of 34 U/mg, caused a bleeding time of 20 min or longer in 70% of the animals after injection of the same number of APTT units, 1,088 U/kg. These data indicate that the APTT activity is a better and more sensitive indicator of the bleeding than is the anti-FXa activity.     

Influence of chemical modification of tryptophan residues on the properties of human antithrombin III

According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.     

Anticoagulant activities and effects on platelets of a heparin fragment with high affinity for antithrombin

A fragment of heparin containing 10–16 sugar units and retaining ability to bind to antithrombin III has been prepared by degrading standard heparin with nitrous acid. This fragment greatly potentiated the inhibition of factor X_a by antithrombin III but had virtually no effect on the inhibition of thrombin. Studies on heparin neutralization showed that the fragment was affected to a much lesser extent than standard heparin by heparin neutralizing components in plasma. The heparin-potentiated aggregation of platelets by low concentrations of ADP was measured for a number of heparin fractions and the fragment. The molecular weight of the heparin was found to be the most important factor determining the platelet aggregation activity, low molecular weight fractions including the fragment being much less active than high molecular weight ones.     

In vivo effects of a low molecular weight heparin fragment on platelet aggregation and platelet dependent hemostasis in dogs

Plasma defibrinogenated dogs were used to study the influence of conventional heparin and a low molecular weight heparin fragment (Fragmin, mean MW 5,000 d) on platelet dependent hemostasis. The heparins were given intravenously in gravimetrically equal doses. The bleeding from standardized skin flap wounds and platelet aggregation (ADP and thrombin) was studied. In comparison, higher doses of the fragment than of heparin were required to increase the bleeding. ADP-induced aggregation in defibrinogenated platelet rich plasma (after addition of normal dog plasma) was potentiated by both heparins. After injection of heparin or the fragment, ADP induced platelet aggregation without prior addition of normal plasma to the test-tube. In conclusion the heparin fragment affected bleeding to a less extent than conventional heparin. One explanation might be a weaker inhibition of thrombin-induced platelet aggregation.     

Influence of tryptophan modification upon digestion of antithrombin III by elastase

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.     

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.     

Influence on the clotting mechanism of sodium pentosan polysulfate (SP54) in comparison to commercial beef lung sodium heparin

Heparin consists of a series of fractions of different molecular weight. Whilst low MW fractions exert a strong anti Xa-activity, high MW fractions mainly enhance thrombin inhibition. A semi-synthetic polysulfated polysaccharide of a MW of 2,000 (Polyanion SP 54) also enhanced the anti Xa-activity. This effect was dose dependent and was comparable to the action of heparin when the substances were tested in a purified system. In plasmatic environment,the anti Xa-activity was lower than the anti Xa-effect of heparin when bovine factor Xa was added to the test system but was less evident when activation of factor Xa was carried out in the test plasma. The antithrombin effect of SP 54 was negligible and was about 4 per cent of the antithrombin effect of heparin. After subcutaneous injection of SP 54,its anti Xa-effect was superior to the effect of a comparable dose of heparin. As a consequence of the low antithrombin potentiating effect,the results of coagulation tests with exception of the aPTT were not influenced by SP 54.     

Distinction of two pathologic antithrombin III molecules: Antithrombin III ‘Aalborg’ and antithrombin III ‘Budapest’

Plasma from two different thrombophilic families with functional inherited antithrombin III deficiency, i.e., with low antithrombin III activity but normal immunoreactive antithrombin III concentration, were investigated simultaneously in the same laboratory. The experiments (thrombin and Factor Xa inactivation, heparin affinity chromatography, modified two dimensional immunoelectrophoresis and gel filtration) showed a distinct difference between the two antithrombin III anomalies. The antithrombin III ‘Aalborg’ had decreased thrombininactivating activity but normal Factor Xa-inactivating activity. The heparin affinity and the molecule weight are normal. The antithrombin III ‘Budapest’ displays a more profound abnormality with pathologic thrombin and Factor Xa inactivation, decreased heparin affinity and abnormal molecular weight.     

Effect of heparin on the vascular distribution of *I-antithrombin III isoforms in rabbits

Studies of radioiodinated antithrombin III kinetic behavior in vivo have shown that a pool of this protein partitions into a vascular compartment that could be the result of antithrombin III interaction with heparin-related glycosaminoglycans present on the vessel wall (J. Clin. Invest. 74, 191-191, 1984). As this partitioning was demonstrated by the rapid initial clearance of the labeled antithrombin III, new studies were performed to determine the effect on this clearance of large doses of intravenously administered heparin. In addition, as previous studies had shown unequal clearance rates for tracer-labeled antithrombin III isoforms differing in affinity for heparin, the effect of heparin treatment was assessed for each isoform. Comparison of data from heparin-treated and control animals demonstrated that heparin markedly decreased the clearance rates for each of the *I-labeled antithrombin III isoforms. Heparin also abolished the difference in *I-antithrombin III isoform plasma-clearance rates, and this effect rapidly disappeared after treatment was discontinued. Inhibition by heparin of the initial clearance of *I-labeled ATIII isoforms is the first direct evidence from in vivo studies for the notion that the pool of antithrombin III which is in rapid equilibrium with the plasma pool is, at least in part, the result of the interaction of antithrombin III with vessel wall heparin-related glycosaminoglycans.     

The mode of action of CY216 and CY222 in plasma.

Three fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and its three fractions the material with high affinity to antithrombin III (AT III) was obtained by chromatography on immobilised AT III. The molecular weight distribution of each of the ten preparations thus obtained was determined by high performance liquid chromatography, while the content of AT III binding material was determined by stoichiometric titration of AT III, monitored by intrinsic fluorescence enhancement. We measured the effect of all heparins on the decay of endogenous thrombin in plasma and on the overall generation of thrombin in plasma, triggered via the extrinsic or via the intrinsic pathway. From these data we calculated the time course of prothrombin conversion, i.e. the course of factor Xa activity as expressed by prothrombinase activity. It was found that in platelet-poor plasma the anticoagulant properties of the heparins are largely dependent on their antithrombin action, which is determined by their content of high affinity material with a MW of 5,400 or higher. The specific antithrombin activity of all heparins, when expressed in terms of material with high affinity to antithrombin III (HAM) with a MW greater than 5,400 is 13.0 min-1/(microgram/ml) (range 10.5-15.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin-induced platelet aggregation. II. Dose/response relationships for two low molecular weight heparin fractions (CY 216 and CY 222)

We have previously shown that unmodified heparin (bovine lung or porcine mucosal) and a low molecular weight heparin fraction, PK 10169, cause platelet aggregation in a dose and molecular weight-dependent manner. In this report, we show that two other low molecular weight heparin fractions, CY 216 and CY 222, also cause platelet aggregation in a dose and molecular weight-dependent manner. Utilizing heparin and defined fractions of CY 216 and CY 222 separated on the basis of molecular weight, we determined dose/response (D/R) relationships for each of these agents and their individual fractions. In comparison to an unmodified porcine mucosal heparin, CY 216 yielded a D/R curve that was shifted down and to the right, indicating that this agent is less potent in causing platelet aggregation. The D/R curve for CY 222, which has a lower molecular weight that CY 216, was shifted further down and to the right, indicating that it was less potent than CY 216. The D/R curves obtained with the fractions of CY 216 and CY 222 demonstrate that as the molecular weight of the fractions decrease, they become progressively less potent in causing platelet aggregation. Fractions with molecular weights of less than approximately 3,000 daltons are essentially without activity in causing platelet aggregation. Platelet aggregation studies with CY 216 and CY 222 fractions separated on the basis of affinity for antithrombin III (AT III) indicate that the platelet aggregating activity of these agents may not be related to their affinity for AT III. However, these latter results are not conclusive and need to be expanded.     

Anticoagulant and antiheparin activities of a pentosan polysulphate

Pentosan polysulphate (PPS, Hemoclar, MW 6,700) was observed to have a low affinity for ATIII-Sepharose eluting at 0.3M NaCl. Tested in vitro it had, as previously reported, a low potency as an anticoagulant, about 10 times less than heparin on a weight for weight basis. Only the KCCT was affected by low concentrations of PPS unlike heparin by which both thrombin time and KCCT were affected. Upon injection of PPS subcutaneously (50mg) the heparin activity measured by chromogenic anti factor Xa and by KCCT was in the ratio of 2:1. When injected intravenously (40mg) into 3 healthy volunteers a significant prolongation of a modified prothrombin time was observed in 2 subjects. When PPS was added to heparin containing plasma it was observed to completely inhibit heparin at low concentrations (2:1 on a weight to weight basis) when measured in the thrombin and prothrombin time but not in the KCCT. The antiheparin effect of PPS was also observed in a purified system in obviating the heparin potentiation of the rate of inhibition of thrombin by antithrombin III. Observations showed that at higher concentrations of PPS it acted by directly inhibiting thrombin without the intervention of antithrombin III but also to potentiate the rate of fibrin monomer polymerization.     

Pharmacological properties of a low molecular weight butyryl heparin derivative (C4-CY 216) with long lasting effects.

We have investigated the pharmacological properties of an O-acetylated butyryl derivative of the low molecular weight heparin CY 216 (C4-CY 216). In a purified system the ability of C4-CY 216 to catalyze thrombin and factor Xa inhibition was comparable to that of CY 216. The antithrombin and antifactor Xa catalytic efficiencies of C4-CY 216 were reduced 217 and 12 times respectively when albumin (10 mg ml-1) was added to the reagents, while those of CY 216 were essentially unchanged. In plasma, the antifactor Xa specific activity of C4-CY 216 was close to that of CY 216 but the antithrombin specific activity was 2 times lower. After bolus and continuous intravenous injection to rabbits, the clearances of the two activities of C4-CY 216 were on average half the corresponding values of CY 216. After subcutaneous injection, the bioavailability of C4-CY 216 was comparable to that of CY 216. C4-CY 216 was as potent as CY 216 in preventing venous thrombosis in the thromboplastin-Wessler model and the duration of the antithrombotic effect was longer than that of the parent compound. The chemical alteration of CY 216 did not enhance the prohaemorrhagic effect in the rat tail transection model. Therefore, the new concept of heparin derivative having a low clearance and long lasting effects that we have recently reported for unfractionated heparin may also be applied to a low molecular weight heparin.     

Pentosan polysulphate: activation of heparin cofactor II or antithrombin III according to molecular weight fractionation

The relative potency of pentosan polysulphate in activation of heparin cofactor II/thrombin interaction has been compared to heparin and dermatan sulphate and found to be within the same order. A skewed distribution of molecular weight forms was observed upon gel filtration of pentosan polysulphate with an average molecular weight of 4500 daltons. Two peaks of activity were observed in activation of heparin cofactor II. The greatest activity was observed in high molecular weight fractions (5-fold greater than that of average molecular weight) and a concentration-dependent profile indicated a template mechanism of action. A lower peak of activity was observed at average molecular weight and the effect of increasing concentrations of this material on activity indicated a mechanism involving binding to proteinase inhibitor or proteinase alone. Potentiation of antithrombin III/thrombin interaction was observed only in fractions greater than the average molecular weight. Concentration-dependent profiles indicated binding to antithrombin III and thrombin was a requisite of activation. A fraction of low molecular weight showed no property of activation of antithrombin III or heparin cofactor II/thrombin interaction. Three fractions of high, average and low molecular weights tested in clotting assays showed relative potencies corresponding to those observed in the purified systems.     

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.     

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.     

The influence of low molecular weight heparin in combination with dihydroergotamine on experimental thrombosis and haemostasis

The influence of low molecular weight heparin in combination with dihydroergotamine (DHE) on thrombus formation and primary haemostasis was investigated in rabbit models. Conventional heparin in the dose of 60 units anti Xa activity/kg b. w. effectively prevented thrombus formation but the same dose of a low molecular weight fragment only gave a marginal decrease of the frequency of thrombosis. The thrombus weight was, however, significantly reduced. Doubling the dose of the heparin fragment totally abolished thrombus formation as did the combination of 60 units with DHE. With DHE it was also possible to diminish the low molecular weight heparin dose to 30 units with a good prophylactic effect. There was a small but significant increase of haemostatic plug formation time in all treatment groups except the one with low molecular weight heparin fragment 30 units anti Xa activity combined with DHE. Thus, by combining low molecular weight heparin in a low dose with DHE it is possible to prevent thrombus formation without influencing primary haemostasis in rabbits.     

A double-blind randomized placebo controlled trial of thromboprophylaxis in major elective general surgery using once daily injections of a low molecular weight heparin fragment (Fragmin)

The safety and efficacy of the low molecular weight heparin fragment (Fragmin) administered as a single daily injection of 2,500 anti Xa units has been evaluated in 183 patients undergoing major elective general surgery. The study was double-blinded and placebo controlled. The active agent, or placebo, was given subcutaneously with the preoperative medication and continued postoperatively for 5-9 days. Ninety five patients received Fragmin and 88 were randomized to receive the placebo. The clinical characteristics of the two treatment groups were similar. Fragmin significantly reduced the incidence of deep venous thrombosis, as detected by a positive 125I fibrinogen leg scan, relative to the placebo treated patients (4/95, 4.2% v. 14/88, 15.9%; p = 0.008). The thrombotic events occurred predominantly (73%) amongst patients with malignancy. Haemorrhagic endpoints necessitating discontinuation of the trial treatment were 4% in each group. No severe adverse reactions or drug related deaths occurred. These results indicate that 2,500 anti Xa units of Fragmin given only once daily is effective thromboprophylaxis for patients undergoing major elective abdominal surgery.     

The molecular mechanisms of heparin action: II. Separation of functionally different heparins by affinity chromatography

Heparin was separated into functionally distinct fractions based on an affinity for bovine antithrombin III or bovine Factor X. Fractions with high affinity for antithrombin III (HA-ATIII) had high activity in the enhancement of antithrombin III inhibition of thrombin. These fractions functioned poorly in the anticoagulation of plasma and as inhibitors of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, HA-ATIII heparin stimulated antithrombin III inhibition of Factor Xa while in the presence of phospholipid HA-ATIII had little effect. Heparin fractions with a high affinity for antithrombin III were poor inhibitors of prothrombin activation and did not inhibit Factor Xa-phospholipid binding.Heparin fractions with high affinity for Factor X(HA-FX) were very effective in the inhibition of prothrombin activation by Factor Xa, calcium and phospholipid, as well as inhibition of Factor Xa binding to phospholipid vesicles. HA-FX fractions of heparin were poor anticoagulants in plasma and did not accelerate inhibition of thrombin by antithrombin III. HA-FX heparin accelerated antithrombin III inhibition of Factor Xa both in the presence and absence of phospholipid.When HA-FX heparin fractions were combined with HA-ATIII heparin fractions, the anticoagulant activity of the mixture was greater than the sum of the activities of the individual fractions. These results indicaté that the interaction of heparin with both Factor Xa and antithrombin III is functionally important in the anticoagulation of blood.