[Three cases of IgM monoclonal gammopathy with haemostasis dysfunction]. / Trois cas de gammapathies monoclonales IgM avec anomalies de l'hémostase.

We relate three cases of IgM gammopathy with haemostatic dysfunction in the perioperative period. The acquired von Willebrand syndrome due to IgM gammopathy is rare and sometimes serious. Its different treatments and their efficiency are discussed: desmopressin, intravenous gammaglobulin, chemotherapy and plasmapheresis.

Pharmacokinetic and antithrombotic properties of two pentasaccharides with high affinity to antithrombin III in the rabbit: comparison with CY216.

This study compares the pharmacokinetic and the antithrombotic properties of two pentasaccharides with high affinity to antithrombin III with those of a conventional low molecular weight heparin, CY216, in the rabbit. On a weight basis, SR 90107A/ORG 31540 (natural pentasaccharide [NPS]) and SR 80027A/ORG 31550 (sulfated pentasaccharide [SPS]) were, respectively, 4.7 and 26 times more potent antifactor Xa inhibitory agents than CY216. They were devoid of antithrombin activity, whereas the antifactor Xa/antithrombin ratio of CY216 was 3.8. After bolus intravenous administration, the clearance (mL/kg/h) of CY216 decreased from 91 +/- 27 for the dose of 12.5 U/kg to 49 +/- 14 for the dose of 50 U/kg and then remained constant up to the highest dose tested (500 U/kg). The clearance of NPS was unrelated to the dose and comparable to that of CY216 over 50 U/kg, whereas that of SPS was 10 times lower. Consistent results were observed after continuous intravenous infusions for 9 hours and subcutaneous administration. The duration of the antithrombotic effect was compared after a single subcutaneous injection of 250 U/kg of either compound in the stasis-Wessler model using human serum as thrombogenic stimulus. Two hours after the injection, the three compounds provided a thrombus prevention of greater than 95% and mean plasma activities of 0.8, 0.9, and 1.9 U/mL for CY216, NPS, and SPS, respectively. Twelve hours after injection, the antithrombotic effects of CY216 and NPS had totally vanished, whereas that of SPS was 68%. At that time, the plasma anti-Xa activities were less than 0.06 U/mL for CY216 and NPS, but 1.1 U/mL for SPS. For the latter compound, significant antithrombotic effects and detectable anti-Xa activities were still recorded 48 hours after the injection. The antithrombotic potency of the three compounds was also compared as their ability to inhibit the growth of a standardized venous thrombosis during 4 hours. The lowest total doses providing the maximum inhibitory effect were 3,125, 1,428, and 62 micrograms/kg for CY216, NPS, and SPS, respectively. These doses generated mean steady state antifactor Xa activities of 1.06, 1.5, and 1.2 anti-Xa U/mL, respectively. These observations indicate that the amplification mechanisms triggered by thrombin bound to fibrin and leading to the generation of new thrombin are essential to ensure venous thrombosis growth and that these mechanisms may be efficiently inhibited by pure antifactor Xa targeting agents.

Pharmacological properties of CY 216 and of its ACLM and BCLM components in the rabbit.

This study compares some in vivo pharmacological properties of CY 216 and of its ACLM and BCLM components having a molecular weight above and below 5.4 kDa respectively. The anti-factor Xa/antithrombin ratio of these compounds determined in a rabbit plasma system were 2.5 and 1.2 for CY 216 and ACLM respectively while BCLM was devoid of anti-thrombin effect. After bolus intravenous injection, continous infusion and subcutaneous administration, the clearances of anti-factor Xa activity generated by ACLM were, on the average, 2 and 1.5 times higher than those generated by BCLM and CY 216 respectively. The clearances of the anti-thrombin activity were comparable for CY216 and ACLM, and higher than those of the antifactor Xa activity. The duration of the antithrombotic effect was investigated in the Wessler model after a single subcutaneous injection of 1000 anti-factor Xa units of one of the compounds. Using thromboplastin as thrombogenic stimulus, the most efficient agent was ACLM and the antithrombotic activity was essentially correlated to the circulating anti-thrombin activity. Using human serum as thrombogenic stimulus, ACLM and BCLM were more efficient than CY 216 and the antithrombotic activity was mainly correlated to the anti-factor Xa activity. The ability of the 3 compounds to inhibit venous thrombosis growth was compared: they were found equipotent and the antithrombotic effect was independent of the anti-thrombin activity. The prohaemorrhagic properties were compared in the rabbit ear model. The activity of the 3 compounds were comparable and significantly less prohaemorrhagic than unfractionated heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II.

The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H-NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.

A comparison of the antithrombotic effects of heparin and of low molecular weight heparins with increasing antifactor Xa/antifactor IIa ratio in the rabbit.

This study compares the ability of unfractionated heparin (UH) and of three low molecular weight heparins (LMWHs) to inhibit venous thrombosis growth in the rabbit. Logiparin (LHN-1), Fraxiparin (CY216) and CY222 were selected because they present very different antifactor Xa/antifactor IIa ratios: 1.7, 3.8 and 6.8 respectively. Heparins were delivered under continuous intravenous infusion for 4 h at increasing doses from 10 to 250 antifactor Xa U kg-1 h-1. The minimum dose providing the maximum inhibitory effect was 50 antifactor Xa U kg-1 h-1. On the basis of this system of units the four heparins were equipotent antithrombotic agents. Due to the highest antifactor Xa/antifactor IIa ratio, CY222 became the most potent antithrombotic agent when the doses were expressed in antithrombin units. Because UH is cleared faster than LMWHs at low dose regimen, the antifactor Xa steady state concentrations generated by the continuous infusion of any of the LMWHs were higher than those generated by UH. In addition the ex vivo antifactor Xa/antifactor IIa ratio of each of the LMWH was superior to the in vitro ratio. These results indicate that the antithrombotic activity of LMWH is not directly related to its subfraction catalysing thrombin inhibition.

Effects of heparin, dermatan sulfate and of their association on the inhibition of venous thrombosis growth in the rabbit.

This study compares the ability of unfractionated heparin, of dermatan sulfate, and of their simultaneous administration delivered as continuous intravenous infusion or as a single bolus injection to inhibit the growth of a standardized venous thrombosis in the rabbit. When delivered as continuous intravenous infusion for 4 h, heparin and dermatan sulfate inhibited thrombus growth in a dose dependent manner. The maximum antithrombotic effect of heparin was achieved at the dose of 0.15 mg kg-1 h-1 (25 U kg-1 h-1) which generated a mean plasma concentration of 1.8 micrograms ml-1 (0.31 U ml-1) and a 1.8 fold prolongation of the activated partial thromboplastin time (APTT) in comparison to the pretreatment value. A comparable antithrombotic effect was obtained with dermatan sulfate at the dose of 2 mg kg-1 h-1. This dose generated a mean plasma concentration of 30 micrograms ml-1 and a 1.3 fold APTT prolongation. Increasing these doses up to 10 fold did not improve the antithrombotic effect which did not overpass 60-70% of the controls. When the compounds were delivered simultaneously, the maximum antithrombotic effect (64%) was obtained with the following association: 0.06 mg kg-1 h-1 (10 U kg-1 h-1) for heparin and 1 mg kg-1 h-1 for dermatan sulfate. Increasing these doses up to 4 to 5 fold did not improve the antithrombotic effect. Heparin, dermatan sulfate and the association of both were also delivered as single bolus injections and the resultant antithrombotic effect was determined 4 h after saline infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of molecular weight upon the anticoagulant and pharmacokinetic properties of dermatan sulfate in the rabbit.

In order to improve the pharmacokinetic properties of unfractionated dermatan sulfate (UDS, mean MW: 25kD), the disposition of 4 low molecular weight dermatan sulfates (LMWDS) with a mean MW ranging from 15 to 4 kD was investigated in the rabbit. In comparison with UDS, it was established that after intravenous administration, the half-life of disappearance, the distribution volume and the clearance of the biological activity increased as the mean molecular weight decreased. After subcutaneous administration, the bioavailability of the 4 LMWDS was improved in comparison with that of UDS, but large inter-animal variations were recorded for LMWDS having a mean MW over 9 kD. Therefore the best compromise between biological activity, clearance, half-life of disappearance, bioavailability and reproducibility after subcutaneous administration should be a compound having a MW ranging from 4 to 9 kD.

Pharmacodynamic properties of long lasting butyryl heparin derivatives in the rabbit.

This paper reports on the pharmacodynamic properties of butyryl derivatives of unfractionated heparin (C4-UH) and of low molecular weight heparin (C4-CY 216) after bolus intravenous injection, constant infusion and subcutaneous administration to rabbits. The pharmacodynamic properties of the two butyryl derivatives were compared to those of the parent compounds, unfractionated heparin (UH) and low molecular weight heparin (CY 216). After bolus intravenous injection of increasing doses, the disposition of the butyryl derivatives were comparable to that of their parent compounds up to 3 mg kg-1. Over this dose, their clearances became 2 to 3 times lower. These long lasting properties were confirmed by constant intravenous infusion experiments. After subcutaneous administration, the bioavailability of C4-UH remained low (10%) at any dose while that of C4-CY 216 ranged from 42 to 120%. If these findings are confirmed in man, these new derivatives open the possibility of treating established deep vein thrombosis with only one daily injection of a butyryl derivative of low molecular weight heparin.

Antithrombotic activity, bleeding effect and pharmacodynamics of a succinyl derivative of dermatan sulphate in rabbits.

This paper compares the pharmacological properties of a new succinyl dermatan sulphate derivative (Suc-DS) to those of the natural dermatan sulphate (DS). Suc-DS was on average 2-3 times more potent than DS in catalysing the inhibition of thrombin by heparin cofactor II and in prolonging the activated partial thromboplastin time and the thrombin clotting time. After bolus injection, Suc-DS was also 2-3 times more potent than DS to prevent experimental venous thrombosis in a Wessler model. Thromboplastin or human serum were used as the thrombogenic stimulus. In contrast, the bleeding effect assessed by rat tail transection technique was comparable. After bolus intravenous injection, the pharmacodynamics of Suc-DS indicated a lower volume of distribution, which was close to the plasma volume, and a slightly lower clearance of elimination. Therefore this chemical alteration of natural DS yields a new compound with an improved antithrombotic benefit/haemorrhagic risk ratio.

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.

Pharmacologic properties of an unfractionated heparin butyryl derivative with long-lasting effects.

This article reports on the pharmacologic properties of an O-acylated butyryl derivative (C4-UH) of unfractionated heparin (UH). In a purified system, the ability of C4-UH to catalyze the inhibition of thrombin and of factor Xa in the presence of antithrombin III was similar to that of UH. Addition of albumin (10 mg/ml) to the reagents reduced the antithrombin and antifactor Xa catalytic potency of C4-UH 68-fold and 20-fold, respectively, and did not alter those of UH. As judged from the prolongation of the activated partial thromboplastin time and the thrombin clotting time, the anticoagulant activities of C4-UH were two times weaker than those of UH. After calibration against UH, the antifactor Xa-specific and antithrombin-specific activities were two and 6.6 times lower, respectively. After bolus intravenous injection into rabbits, the apparent clearances of C4-UH were reduced 2.4 (antifactor Xa activity) and 3.2 times (antithrombin activity) in comparison with those of UH. This property accounted for the higher plasma concentrations generated during a constant infusion of the same dose. In the Wessler thromboplastin model, the minimum doses providing the maximum antithrombotic effect after bolus injection were equivalent for both compounds when expressed as antifactor Xa units; the duration of the antithrombotic effect of this derivative was prolonged, whereas the hemorrhagic potential was unaffected. This study opens a new concept for heparin derivatives having lower clearances and long-lasting effects. These properties could be linked to nonspecific binding of C4-UH to plasma proteins, thereby reducing the amount of free compound available to interact with antithrombin III.

Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit.

Our purpose was to determine the relative contribution of the antifactor Xa and antithrombin activities of heparin to its antithrombotic potency. The antithrombotic activities of unfractionated heparin (UH), two low molecular weight heparins (LMWH, CY 216 and CY 222) with increasing anti-factor Xa/antithrombin ratio and a synthetic pentasaccharide (PS) with high affinity to antithrombin III and no antithrombin activity were evaluated. In the Wessler-thromboplastin model, the most potent antithrombotic agent, on a weight basis, was UH followed by CY 216, CY 222 and the PS which was 40 times less potent than UH. On an antithrombin unit basis, the antithrombotic potencies of UH, CY 216 and of CY 222 were equivalent. Thus, in this model, the antithrombotic effect results from the catalytic action of UH or LMWH on thrombin inhibition. In the Wessler-serum model, on a weight basis, the antithrombotic effectiveness of UH was unchanged, those of CY 216 and CY 222 were doubled, and that of the PS was increased 10 times. On an anti-factor Xa unit basis, CY 216 was as effective as UH, and PS as effective as CY 222. On an antithrombin unit basis, CY 216 and CY 222 were equivalent and more potent than UH. Thus, in this model, the antifactor Xa activity of heparin becomes important for its antithrombotic property. After a single subcutaneous injection of 1000 antifactor Xa U/kg, the antithrombotic effects of UH were maintained for more than 14 h in the two models. After injection of the same dose of CY 216 significant antithrombotic effects were observed only for 9 h, in the Wessler-thromboplastin model but for 18 h in the Wessler-serum model. At that time, no detectable antithrombin activity was measurable in the plasma while 0.11 units of antifactor Xa activity/ml was detected. Thus, the relative contribution of the anti-factor Xa and antithrombin activities to the antithrombotic effect of a LMWH differs according to the nature of the thrombogenic stimulus.

Pharmacokinetics of heparin and low molecular weight heparin.

After parenteral injection, heparin is removed from the blood via two mechanisms, saturable and non-saturable. The saturable mechanism represents clearance by the reticuloendothelial system and endothelial cells, to which heparin binds with a high affinity. The non-saturable mechanism is represented by renal excretion. The contribution of the two mechanisms to the clearance of heparin varies according to the dose delivered and the molecular weight of the heparin preparation. At low doses, unfractionated heparin (UH) is removed mainly via the saturable mechanism, while at higher doses the contribution of the non-saturable mechanism to its clearance becomes pre-eminent. This model accounts for the major pharmacokinetic properties of UH. After bolus intravenous injection of low doses, UH disappears from the blood exponentially with a dose-dependent half-life; at higher doses, UH disappears with a concave-convex pattern. Under continuous intravenous infusion there is a non-linear relationship between the dose of UH injected and the steady-state plasma concentration. After subcutaneous injection, the bioavailability of the anti-factor Xa activity increases with the dose delivered and tends toward 100% at high doses. In contrast, low molecular weight heparins (LMWH) are mainly removed by non-saturable renal excretion. This explains the dose independence of the pharmacokinetic parameters of LMWH, the excellent bioavailability of the subcutaneous route at any dose, and the prolongation of LMWH half-life in cases of chronic renal insufficiency. However, the model does not explain the large interindividual variability of the pharmacokinetic parameters of both UH and LMWH.

Pharmacologic properties of a low molecular weight dermatan sulfate: comparison with unfractionated dermatan sulfate.

The anticoagulant, pharmacodynamic, and antithrombotic properties of a low molecular weight dermatan sulfate (molecular weight range 1600 to 8000, peak 4000) were compared with those of unfractionated dermatan sulfate (molecular weight range 12,000 to 45,000, peak 25,000). Anticoagulant activities were evaluated as the ability of the compounds to catalyze the inhibition of thrombin in the presence of heparin cofactor II in a purified system and to prolong the activated partial thromboplastin time or the thrombin clotting time of human and rabbit plasmas. On the basis of weight, low molecular weight dermatan sulfate was two times less potent than unfractionated dermatan sulfate. After bolus intravenous injection into rabbits, the volume of distribution of low molecular weight dermatan sulfate was 10 times larger than that of unfractionated compound, and the half-life of disappearance was two to four times longer despite a 1.4 to 2.3 times higher total clearance. The bioavailability of low molecular weight dermatan sulfate from its subcutaneous depot was 100%; it was absorbed faster from that depot than unfractionated dermatan sulfate. The antithrombotic activities of unfractionated and of low molecular weight dermatan sulfate were also examined with a Wessler-type model with tissue factor as the thrombogenic stimulus. When evaluated 3 minutes after a bolus intravenous injection, unfractionated dermatan sulfate was twice as active as low molecular weight dermatan sulfate on the basis of weight. With subcutaneous injection, 10 mg/kg of low molecular weight dermatan sulfate generated an activity in plasma equivalent to 5.6 micrograms/ml 1 hour later. This concentration was associated with a significant antithrombotic effect that lasted for less than 6 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Unfractionated heparin and CY 216: pharmacokinetics and bioavailabilities of the antifactor Xa and IIa effects after intravenous and subcutaneous injection in the rabbit.

This report compares the pharmacokinetics and the bioavailabilities of the antifactor Xa and of the antifactor IIa activities generated by intravenous (IV) and subcutaneous (SC) injections of increasing doses of unfractionated heparin (UH) and of a low molecular weight heparin (CY 216). Rabbits were injected with 500, 1,000, 2,500, and 5,000 antifactor Xa u/kg of both heparins and their biological activities were followed at various time intervals. After IV injection the clearance of the antifactor Xa activities was independent of the dose and the clearance of UH was significantly higher than that of CY 216; after SC injection the bioavailability estimated from the antifactor Xa effect was consistently over 100% for CY 216 while that of UH increased from 27% at the lowest dose to 93% at the highest dose. The pharmacokinetic parameters estimated by the antifactor IIa activity of UH were superimposable to those calculated with the antifactor Xa activity. For CY 216 no direct comparison between the two activities was made since the dose injected expressed in antifactor IIa units was 3.4 times lower. UH and CY 216 were therefore injected intravenously to other animals at equivalent and increasing doses expressed in antifactor IIa units (50-5,000 u/kg). The pharmacokinetic parameters calculated from the curves of the antifactor IIa activities were basically identical except at the two lower doses (50 and 100 u/kg) for which UH was cleared faster than CY 216.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutralization of dermatan sulfate in vitro and in vivo by protamine sulfate and polybrene.

The neutralization of the anticoagulant, anti-thrombin, and bleeding effects of dermatan sulfate (DS), a potential antithrombotic agent, was investigated. Protamine sulfate (PS) and hexadimethrine bromide (Polybrene), which reverse the anticoagulant effect of heparin, also neutralized DS in vitro. In human plasma, polybrene was approximately 3 times more active on a weight basis than PS for neutralizing DS (1.5 micrograms polybrene inhibits 1 microgram DS). Intravenous administration of polybrene to rabbits pretreated with DS in a 1:1 weight ratio immediately neutralized 90% of DS and this effect was stable with time. In contrast, PS in a weight ratio of 6:1 (PS to DS) only neutralized 50% of DS injected. When plasma DS concentrations were maintained by continuous infusion between 3 and 15 micrograms/ml, a bolus of polybrene 0.25 mg/kg induced an immediate drop of about 4 micrograms/ml but initial values of DS were recovered within 20 min. PS was again much less effective than polybrene for neutralizing DS. The bleeding effect of DS and its correction by polybrene was studied by using the rat tail transection model. Very large doses of DS (greater than 10 mg/kg) were required to get a modest prolongation of bleeding time. The injection of equivalent doses of polybrene in animals pretreated by DS induced a strong bleeding effect associated with a drop in platelet and leukocyte counts. Animal models are thus inappropriate for investigating the correction of DS-induced bleeding, because high doses of both DS and neutralizing agents are required in these models. Our results indicate that, provided the doses of neutralizing agents remain below their established levels of toxicity in man, DS could if necessary be neutralized completely by polybrene and partially by PS.

Pharmacokinetics of heparin and related polysaccharides.

The pharmacodynamic profile of standard heparin (SH), a low molecular weight derivative (CY 216) and of dermatan sulfate (DS), a new potential antithrombotic drug, was investigated in the rabbit over a large range of doses. After bolus i.v. injection of low doses, the biological activity of SH disappeared exponentially; however, its half-life was prolonged when the dose injected increased, and over 158 micrograms/kg (100 anti-factor Xa U/kg) the biological activity disappeared as a concave-convex curve. CY 216 disappeared more slowly than SH at low doses but faster than SH at higher doses. More than 90% of the DS biological activity present 1 minute after the i.v. injection disappeared exponentially without dose-dependent effects. Increasing doses of the three drugs were then delivered for 5 h under continuous infusions. Below 500 micrograms/kg/h the DS and CY 216 plateau concentrations were higher than that of SH while above this dose the SH concentration was higher than that of DS and CY 216. These observations may be explained by the results of pharmacokinetics experiments where 125I-labeled compounds were delivered by bolus i.v. injection in association with increasing doses of their unlabeled counterparts. For SH there was a 10-fold difference between the half-life of the lower dose (32 micrograms/kg or 5 anti-factor Xa U/kg) and that of the higher dose (3200 micrograms/kg); it was demonstrated that the half-life of SH continuously shortened as its plasma concentration decreased. In contrast the CY 216 and DS half-lives were very close, independent of the dose delivered, and therefore longer than that of SH at low doses and shorter than that of SH at higher doses. The renal contribution to the clearance of SH, CY 216, and DS was also investigated by injecting a low dose (150-200 micrograms/kg) and a 10 times higher dose to sham-operated or binephrectomized animals: renal function is critical for the elimination of the three compounds from the blood except for SH at low doses. These observations are promising for the development of DS and may have clinical implications.