Discordance between the anti-Xa activity and the antithrombotic activity of an ultra-low molecular weight heparin fraction

The relationship between the in vivo antithrombotic effect of heparin and ex vivo anti-Xa activity has been investigated using an animal thrombosis model. Three low molecular weight heparins were compared with the standard heparin from which they were fractionated. All four heparins showed a dose-dependent antithrombotic effect enabling the relative antithrombotic and anti-Xa activities to be compared over a dosage range. A correlation between ex vivo anti-Xa heparin levels and antithrombotic effect was demonstrated for the standard (MW 16,000), intermediate (MW 7,600) and low (MW 4,600) molecular weight heparins but not for the ultra-low molecular weight (MW 3,000) fraction. The lack of relationship between anti-Xa activity and inhibition of thrombosis for the very low molecular weight fraction indicates that a very high anti-Xa activity (measured in vitro or ex vivo) is not always predictive of in vivo antithrombotic efficacy. These findings suggest that other properties of low molecular weight heparins contribute to their antithrombotic effectiveness.     

Comparison of the in vivo hemorrhagic and antithrombotic effects of a low antithrombin-iii affinity heparin fraction

Antithrombin III (ATIII) affinity chromatography of commercial grade heparin yields fractions of high and low affinity for ATIII. In vitro, the high affinity fraction accounts for most of the anticoagulant effect while there is evidence that the low affinity material interferes with platelet function. We have studied the relative antithrombotic and hemorrhagic effects of low affinity heparin. The low affinity heparin fraction, specific activity 43 USP units/mg, was compared with standard heparin (150 USP units/mg) in rabbit experimental models. A residual 12.5% by weight of this low affinity heparin showed high affinity for ATIII. Inhibition of thrombosis in a stasis-hypercoagulability model was directly related to the weight (mg) of high affinity material in each of the heparins. In the bleeding model, when similar weights (mg) of high affinity material were infused, significantly more bleeding was demonstrated with the low affinity fraction which contained a 5-fold excess by weight of low affinity material. We have demonstrated that a low affinity heparin depleted of in vitro anticoagulant and in vivo antithrombotic activity significantly contributes to hemorrhage.     

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

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

Subcutaneous treatment of deep venous thrombosis with low molecular weight heparin. A dose finding study with LMWH-Novo

Treatment of deep venous thrombosis with low molecular weight heparin (LMWH-Novo, Logiparin) was carried out with two different doses of Logiparin, 75 XaI U/kg b.w. twice daily and 150 XaI U/kg b.w. once daily subcutaneously for 5 days. Simultaneously warfarin was given from the first day of heparin treatment. Mean age of the twenty patients was 65 years and one third was females. No serious side effects, hematomas, pulmonary emboli or signs of recurrent thrombosis occurred during treatment with either dose regime. Venografic assessment with Marder scoring one week after initiation of Logiparin treatment showed a slight not significant improvement apparent in 40 % of the patients. The activities of F-IIaI and F-XaI in the blood plasma were found to increase after injection of Logiparin. These two parameters seem to be the most suitable for monitoring the effect during treatment. For future studies on the therapeutic effect of Logiparin in deep venous thrombosis a single dose of 150 to 200 F-XaI activity per 24 hours seems to be most suitable.     

A comparison of the anti thrombotic and haemorrhagic effects of low molecular weight heparin fractions: The influence of the method of preparation

Bleeding is an important complication of heparin therapy. A number of low molecular weight heparin fractions produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. Low molecular weight heparin fractions and fragments are produced by a number of different procedures but their relative effects on haemostasis and thrombosis have not been evaluated. We have compared the antithrombotic and haemorrhagic effects of two low molecular weight heparin fragments and of a heparinoid with porcine mucosa heparin and related these in vivo findings to the results of ex vivo tests of blood coagulation and in vitro tests of platelet function. Haemorrhage was assessed using a rabbit ear bleeding model. The antithrombotic effects were assessed by measuring inhibition of a tissue thromboplastin-induced jugular vein thrombus and by inhibition of fibrin and platelet accumulation in an arterial-venous shunt. The ex vivo anticoagulant effects were     

Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate.

In general, the in vitro anti Xa activity of low molecular weight heparins is neutralized to a lesser degree than the anti Xa activity of unfractionated heparin. To determine whether these differences occur in vivo, a rabbit stasis thrombosis model and a rat laser-induced thrombosis model were utilized. In the laser model, a similar degree of neutralization of the antithrombotic activity of heparin and Fraxiparin was obtained. However, in the stasis thrombosis model, significant antithrombotic activity of Fraxiparin remained after equigravimetric protamine administration. Ex vivo APTT, thrombin time, Heptest, amidolytic anti Xa and anti IIa assays were performed. A coefficient (r = .806) was obtained for the correlation of Heptest activity to antithrombotic effect in the stasis thrombosis model, while the coefficients obtained for the other tests ranged from .152-.570. However, after neutralization by protamine, the thrombin time exhibited the highest correlation coefficient (r = .685) between ex vivo activity and residual antithrombotic effect. Since Fraxiparin retains antithrombotic activity after protamine administration, clinical benefit may be observed for this low molecular weight heparin as compared to unfractionated heparin after neutralization.     

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.     

Anticoagulant and antithrombotic effects of combinations of defibrotide with heparins and other antithrombotic agents in an animal thrombosis model

Defibrotide, acetylsalicylic acid, low molecular weight heparin (CY 216), sodium heparin, Molsidomin and a thromboxane receptor antagonist have been investigated separately and in combinations in an animal thrombosis model in which rat mesenteric venules are damaged by defined laser energy. Furthermore, the ex vivo anticoagulant activity of Defibrotide and heparin were studied in rat plasma. All investigated agents showed a significant and dose-dependent antithrombotic effect when venules were damaged. A strong additive effect was observed when Defibrotide was administered together with heparins. The combinations of Defibrotide with other antithrombotic agents did not have significant additive effects. In the present study the combination of Defibrotide and heparins showed a prolongation of aPTT and Heptest in comparison with heparin given exclusively.     

Effects of an enzymatically depolymerized heparin as compared with conventional heparin in healthy volunteers

A low molecular weight heparin (LMW-heparin) with a mean molecular weight of 4900 dalton was prepared by controlled enzymatic depolymerization of conventional porcine mucosal heparin. The effects of 2,500, 5,000 and 10,000 U (XaI; 29, 58 and 116 mg) on factor Xa inhibition (XaI), factor IIa inhibition (IIaI), APTT, AT III and platelet count were compared to those of 5,000 U (XaI; 26 mg) of conventional heparin given s.c. to 6 healthy volunteers. 5,000 U (XaI; 58 mg) of LMW-heparin was given i.v. A dose related response with regard to the XaI and the IIa-inhibitory activities with peak values at 4 hours after the s.c. injections was obtained. An increase of the XaI/IIaI ratio over the time after injection was seen only after i.v. administration of the LMW-heparin. The APTT was only slightly prolonged and remained within normal range after s.c. injection. AT III and platelet counts were unaffected. The biological half life of the LMW-heparin was 111 minutes if assayed by Xa inhibition, 76 minutes if assayed by IIa inhibition and 40 minutes if assayed by APTT. A strong correlation between the XaI activities obtained and body weight was seen, indicating that LMW-heparin should be administered individually according to body weight.     

A comparison of the antithrombotic and haemorrhagic effects of a low molecular weight heparin (LHN-1) and conventional heparin

The antithrombotic effects after intravenous administration of a low molecular weight heparin (LHN-1) and conventional heparin were compared in a rabbit model of experimental thrombosis, where thrombus formation was induced by a combination of endothelial damage and stasis. Both compounds were able to prevent thrombosis completely. However, LHN-1 was significantly less potent than conventional heparin, the ratio between doses with the same antithrombotic effect being 2.4:1 on a weight basis. Bleeding times after administration of LHN-1 and conventional heparin were determined by tail transsection in anaesthetized rats and by template bleeding in the ear of conscious pigs. Given intravenously at a dose ratio of 2.4:1 (w/w), LHN-1 affected APTT less than conventional heparin, whereas the effects on haemostasis were not significantly different. In conclusion, it was found that after intravenous administration LHN-1 prevented experimental thrombosis as effectively as conventional heparin. However, the correlation between antithrombotic and haemorrhagic effects of LHN-1 was the same as that of conventional heparin. The corresponding relation in man remains to be determined.     

Anticoagulant effects of two types of low molecular weight heparin administered subcutaneously

Two types of LMW heparin were prepared by gel filtration of standard heparin (LMW fraction) and by degradation of heparin by nitrous acid (LMW fragment), respectively. The effects on factor Xa inhibition (XaI), APTT, platelet aggregation and AT III level of these preparations were studied after subcutaneous administration to humans and compared with those of standard heparin. At a dose of 5000 IU (XaI) the LMW fraction and LMW fragment induced peak plasma XaI activity of 0.32 IU/ml and 0.41 IU/ml respectively, compared to 0.07 IU/ml for heparin. Still 11.5 h after administration both LMW preparations gave higher activities than heparin ever induced. Following administration of 10,000 IU (XaI) of the LMW fragment the plasma peak XaI activity was 0.81 IU/ml. This prolonged the APTT from 36 sec to 46 sec only. The half-lives of the XaI activity in plasma were between 3 and 4 hours. No effect on platelet aggregation or AT-III level was demonstrated.     

Neutralization of a low molecular weight heparin (LHN-1) and conventional heparin by protamine sulfate in rats

The neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized. In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.     

Low molecular weight heparin and the heparin mobilisable pool of platelet factor 4 are reduced in young survivors of myocardial infarction

The platelet factor 4 (PF4) mobilisation properties of low molecular weight heparin (Fraxiparine, Sanofi Winthrop, France) in young survivors of myocardial infarction (YSMI) and healthy volunteers have been investigated. The study group consisted of 42 YSMI less than 44 years old, all of them with angiographically proven occlusive coronary artery disease, studied 6 to 24 months after the acute event. The control group was composed of 30 healthy men of similar age. Subjects from the study and control groups were allocated to the following subgroups: those receiving 60 or 120 IU/kg b.w. of standard heparin (Polfa Kutno, Poland) and those receiving 60, 120 or 180 IC anti-Xa U/kg b. w. of low molecular weight heparin (Fraxiparine, Sanofi Winthrop, France) as a single intravenous injection. Additionally, in five YSMI patients the influence of prolonged aspirin administration (0.3g daily for more than 30 days) on the Fraxiparine mobilisable pool of PF4 and β-thromboglobulin (β-TG) concentration in the plasma was determined after injection of 180 IC anti-Xa U/kg b.w. of the drug. The PF 4 and β-TG concentration in the plasma was evaluated using enzyme immunoassay methods before heparin or Fraxiparine intravenous injection and 2, 5, 10, 20, 30 and 60 min after. In both, the control and YSMI groups baseline PF4 levels were found to be normal. Moreover, similar marked dose-dependent increases of PF4 concentration in the plasma measured after 60 and 120 IU/kg b.w. of heparin as well as after 60 and 120 IC anti-Xa U/kg b.w. of Fraxiparine was found. The administration of 120 IU/kg b.w. of heparin resulted in a reduced rise in plasma PF 4 in YSMI as compared to healthy controls. The same phenomenon was observed when 180 IC anti-Xa U/kg b. w. of Fraxiparine was injected intravenously. In YSMI treatment with aspirin had no influence on the Fraxiparine mobilisable pool of PF 4 or the β-TG concentration in the plasma. These results suggest that mobilisable pool of platelet factor 4 in young survivors of myocardial infarction derives from the “nonplatelet pool” and that reduction of heparin- or Fraxiparine-releasable pool of PF4 may reflect an impaired endothelium function, probably due to atherosclerosis.     

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.     

Ex vivo activity of heparin is not predictive of blood loss after neutralization by protamine.

To study whether the ex vivo activity of heparin and Fraxiparin correlates to and predicts the extent of blood loss induced by the heparins (pre- and post neutralization by protamine), a rat tail transection model and a rabbit ear bleeding model were used. In the rat model heparin (2 mg/kg i.v.) significantly prolonged the bleeding time, while this dose of Fraxiparin had no effect. In the rabbit ear blood loss model, heparin (2 mg/kg i.v.) produced significant increases in blood loss while Fraxiparin (2 mg/kg i.v.) produced approximately 30% of the blood loss induced by heparin. Equigravimetric protamine reduced the heparin-induced blood loss by approximately 50%, however, significant blood loss, thrombin time and Heptest activity remained. Heparin and Fraxiparin (3 mg/kg s.c.) did not cause any increased bleeding. While, all activities of heparin were completely neutralized by protamine, the Heptest activity of Fraxiparin was resistant to neutralization. The ex vivo activity of heparins after neutralization by protamine does not correlate to the extent of blood loss which suggests it may not be necessary to neutralize all ex vivo activities of the heparins to baseline values to be assured that blood loss is reversed.     

The effect of unfractionated vs. low molecular weight heparin on tissue factor pathway inhibitor levels in hospital inpatients.

Although heparin is widely used as an antithrombotic agent, its multiple mechanisms of action are not fully defined. Recent work has suggested that tissue factor pathway inhibitor (TFPI) may contribute to the antithrombotic activity of heparin by inhibiting the extrinsic pathway of coagulation. We have investigated the effect of heparin on TFPI and have found that when unfractionated heparin is given by continuous intravenous infusion to hospitalized inpatients, TFPI levels increase 2.3-fold and remain high as long as heparin is continued, but return to baseline levels soon after the infusion is stopped. In contrast, therapeutic doses of the low molecular weight heparin, dalteparin, resulted in significantly less TFPI induction. Given the increasing number of studies establishing the clinical efficacy of low molecular weight heparins as antithrombotic agents, these results suggest that TFPI may not be a major contributor to the antithrombotic effect of heparin.     

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".     

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.     

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.     

Plasma levels of total and free tissue factor pathway inhibitor (TFPI) as individual pharmacological parameters of various heparins

The release of circulating tissue factor pathway inhibitor (TFPI) into plasma by heparins is thought to contribute to their overall antithrombotic activity. In the presented study in healthy volunteers, we measured the heparin-induced increase of circulating total and free TFPI antigen and the aXa- and aIIa activity after subcutaneous (s.c.) injection of 9000 aXa-U of four different heparins: unfractionated heparin (UFH) (13.0 kDa), a medium molecular weight (MW) heparin with a narrow MW range (HF) (10.5 kDa), certoparin (6.0 kDa) and enoxaparin (4.5 kDa). Based on the administration of equi-active aXa doses, certoparin induced the highest increase in total TFPI determined as AUC (p <0.01). The lowest effect was observed for UFH (p <0.0001). However, the AUC of released free TFPI significantly increased in the order: enoxaparin < UFH < certoparin < HF, showing MW dependency with the exception of UFH. Comparing the effects of equi-gravimetric heparin doses, the MW dependency becomes even more pronounced. The mismatch of UFH may be due to its poor bioavailability, which becomes obvious from its low ex vivo aXa activity. In contrast to the TFPI releasing potency, the ex vivo aXa activity continuously decreased with increasing MW. Although the ex vivo aIIa activity of the heparins increased in the same order like the release of free TFPI, there was no clear correlation. This is attributed to the fact that the aIIa activity of heparin is not only dependent on the MW, but, in contrast to its TFPI releasing effect, also on the percentage of material with high affinity to AT. In conclusion, besides the aXa- and aIIa activity, the TFPI releasing effect of heparins is an additional parameter of their individual pharmacological profile.