Relationship between ex vivo anti-proteinase (factor Xa and thrombin) assays and in vivo anticoagulant effect of very low molecular weight heparin, CY222

There is uncertainty as to which activities of unfractionated heparin (UFH) and low MW heparin are responsible for their anticoagulant and antithrombotic properties. We have sought to answer this question by examining plasma samples taken during a recently conducted dose-finding study of the low MW heparin, CY222, in haemodialysis for chronic renal failure. In this study, in vivo anticoagulant effect was assessed by measurement of plasma FPA levels. UFH was administered as a dose of 5000 iu bolus + 1,500 iu/h maintenance infusion, while the effects of three doses of CY222 were studied (10,000, 15,000 and 20,000 Institute Choay anti-factor Xa u bolus, all with 1,500 Institute Choay anti-factor Xa u/h maintenance infusion). Anti-factor Xa levels were determined by chromogenic substrate assay. Anti-thrombin levels were determined by chromogenic substrate assay and by quantitation of catalysed thrombin-inhibitor complexes (using autoradiography). Analysis of the results indicate that plasma fibrinopeptide A (FPA) levels correlate with anti-factor Xa (r = -0.45) and anti-thrombin (substrate) (r = -0.63) levels of UFH, but only with the anti-factor Xa levels (r = -0.41) of CY222. These results suggest that the anti-factor Xa assay is currently the most suitable assay for monitoring low MW heparins such as CY222 in humans.     

The importance of thrombin inhibition for the expression of the anticoagulant activities of heparin, dermatan sulphate, low molecular weight heparin and pentosan polysulphate

The effects of standard heparin, three low molecular weight derivatives of heparin, dermatan sulphate and pentosan polysulphate on the intrinsic coagulation pathway were compared in order to evaluate the contributions of the anti-factor Xa and anti-thrombin activities to their anticoagulant activities. The anticoagulant potency was measured by the ability of each sulphated polysaccharide to inhibit the generation of thrombin activity in plasma. Similarly, the ability of the six sulphated polysaccharides to enhance the rates of inactivation either factor Xa or thrombin in defibrinated plasma containing calcium chloride and cephalin were also determined. Standard heparin was the only sulphated polysaccharide that could equally inhibit thrombin generation and enhance the inactivation of factor Xa and thrombin by plasma. Dermatan sulphate and pentosan polysulphate were more effective as inhibitors of thrombin generation than potentiators of factor Xa inactivation. The two smallest derivatives of heparin, which had high anti-factor Xa (but low antithrombin) activity, were the poorest inhibitors of thrombin generation. Our results therefore suggest that only sulphated polysaccharides that enhance the inactivation of thrombin by plasma and/or inhibit the generation of thrombin activity in plasma are good anticoagulants. These two activities of sulphated polysaccharides appear to be good predictors of the relative antithrombotic potency in vivo.     

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.     

The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin

The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.     

Placental transport of low molecular weight heparin in the pregnant sheep

Standard heparin, an effective treatment for antepartum thromboembolic disease, is thought to be safe for the fetus since it does not cross the placenta. Recently, a number of low molecular weight heparins have been prepared which have been shown to produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. These observations suggest that the low molecular weight heparins may also provide superior antithrombotic therapy in antepartum thromboembolic disease. However, it is not known whether the low molecular weight heparins cross the placenta. To determine this, we examined the pharmacokinetics of 125I-labelled standard heparin and a low molecular weight heparin, and their anticoagulant effects in mother and fetus, using a pregnant sheep model. Catheters were inserted into maternal and fetal femoral arteries at 108-119 d gestation (term: 147 d). 1-3 days later the mothers were given a bolus i.v. injection of 5000 anti-Xa units of 125I-labelled standard heparin or low molecular weight heparin, CY 222. Nine serial blood samples were collected over 4 h from both mother and fetus for measurements of radioactivity, anti-Xa activity (chromogenic) and activated partial thromboplastin times. When therapeutic levels of standard and CY 222 heparins were achieved in the mother, there was no detectable radioactivity or anticoagulant effect in the fetus. We conclude that standard heparin and the low molecular weight CY 222 do not cross the placenta in the pregnant sheep.     

Comparative study on a new one-stage clotting assay for heparin and its low molecular weight derivatives

The effects of a normal and a low molecular weight (LMW) heparin fraction were compared by four coagulation methods. Plasma samples of patients were investigated who were treated with normal heparin or with a LMW heparin. The study was undertaken to analyze the interrelationship between the coagulation methods: Heptest, activated partial thromboplastin time, thrombin clotting time, and S 2222 chromogenic anti-factor Xa test. The results showed a high correlation between Heptest and S 2222 anti-factor Xa method for unfractionated and LMW heparin (r = 0.91 and 0.90). Comparing the coagulation times in seconds of Heptest and aPTT, the correlations were r = 0.56 (normal heparin) and 0.15 (LMW heparin). The correlation between the coagulation times of Heptest and thrombin clotting time were r = 0.65 and 0.25, respectively. The correlations between the coagulation methods were higher, when coagulation times rather than transformed values to units per liter of the Heptest and of the S 2222 anti-factor Xa method were employed. Furthermore, the data demonstrate a high sensitivity of the Heptest to conventional and LMW heparin, whereas activated partial thromboplastin time and thrombin clotting time are less sensitive to either heparin. For laboratory control of LMW heparin, Heptest and S 2222 chromogenic method are reliable tests. Therapeutic ranges will have to be established.     

Comparative human pharmacology of low molecular weight heparins

The pharmacodynamics of LMW heparins differ consistently from those of normal heparin. The relative bioavailability is greater than 90% and for some LMW heparins more than 100% after subcutaneous administration, as measured by the anti-Factor Xa activity. This indicates release of endothelial bound glycosaminoglycans with anti-Factor Xa-like activities. Direct determination of the LPL and HTGL activities demonstrate higher capacities of two and a lower capacity of three LMW heparins to release these enzymes from their endothelial glycoprotein receptors into the bloodstream. Thus, all LMW heparins are characterized by improved pharmacodynamics compared with normal heparin, but their anti-Xa to APTT and anti-Xa to anti-IIa ratios and half-lives differ among each other. The neutralization of the anticoagulant effects of LMW heparins by protamine are similar in vitro and in vivo at equigravimetric doses. However, unfractionated heparin and LMW heparins are not completely antagonized by protamine on a whole blood clotting test (thrombelastography). This inhibition of Factor Xa of LMW heparins is counteracted not completely by protamine. This may be due to a release of other glucosaminoglycans by LMW heparins. The present overview demonstrates that a comparative human pharmacology gives useful devices for prophylactic and therapeutic regimen for LMW heparins in patients in addition to the data obtained from animal models.     

Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.     

Low molecular weight heparins – Pharmacological principles and indications in clinical practice

Summary The pharmacological effects of low molecular weight heparins are characterized by an increased inhibition of factor Xa and decreased inhibition of thrombin of the coagulation cascade in comparison to unfractionated heparins. After administration into the circulation blood, levels are sustained twice as long as for unfractionated heparin on factor Xa inhibition leading to a biological half-life of 2 h after intravenous administration and of 4 h after subcutaneous administration. The clearance of LMW heparins is decreased and the area under the activity time curve is increased more than 2-fold compared to unfraction-ated heparin. The effects on bleeding time and hemorrhage are decreased in animal models. In postoperative medicine LMW heparins once daily (o.d.) have a higher efficacy as low dose unfractionated heparin three times daily (t.e.d.). In medical bedridden hospitalized patients LMW heparin o.d. is as effective and probably safer than unfractionated heparin t.e.d. Heparin-induced thrombocytopenia type II occurs less frequently. Treatment of recent deep venous thrombosis is performed more effectively and safely using low molecular weight heparins. So far, there is no sufficient evidence for different efficacies of LMW heparins in these indications. The efficacy and safety of low molecular weight heparins is currently proven in patients with cerebral ischaemia and unstable angina.     

Is impaired renal function a contraindication to the use of low-molecular-weight heparin?

BACKGROUND: Because of the risk of accumulation of anticoagulant effect, it has been suggested that patients with a creatinine clearance of 30 mL/min or less (< or =0.50 mL/s) should be excluded from treatment with low-molecular-weight (LMW) heparin, or have anti-factor Xa heparin level monitoring performed. OBJECTIVE: To assess the appropriateness of this recommendation. METHODS: We performed a systematic search of MEDLINE, EMBASE, and International Pharmaceutical Abstracts to identify prospective articles comparing differences in the pharmacokinetics of LMW heparins in nondialyzed patients with varying degrees of renal function. Reference lists of retrieved reports were checked for additional articles. RESULTS: Three single-dose pharmacokinetic trials and 2 multiple-dose deep vein thrombosis (treatment trials met our selection criteria. The 3 trials that could address our primary objective did not support the use of a 30-mL/min (0.50-mL/s) cutoff of creatinine clearance to select individuals at risk of accumulation when LMW heparin is used. Four of the 5 trials support the notion that anti-factor Xa activity of some LMW heparin preparations accumulates in patients with impaired creatinine clearance. Tinzaparin sodium, an LMW heparin with a higher-than-average molecular weight distribution, appears to be the exception, since it did not exhibit accumulation in patients with creatinine clearances as low as 20 mL/min (0.33 mL/s). CONCLUSIONS: The use of a 30-mL/min (0.50-mL/s) cutoff is not justified, on the basis of currently available evidence, to select individuals at increased risk of accumulation when LMW heparin is used. The pharmacokinetic response to impaired renal function may differ among LMW heparin preparations.     

Determination of the levels of unfractionated and low-molecular-weight heparins in plasma: their effect on thrombin-mediated feedback reactions in vivo. Preliminary results after subcutaneous injection.

We define a standard independent unit (SIU) of heparin as that amount that, in plasma containing 1 mumol of ATIII, raises the (pseudo-)first-order breakdown constant of factor Xa by 1 min-1. These units measure all material with a high affinity for ATIII (HAM); only material above the critical chain length of 17 monosaccharide units (above critical chain length material; ACLM) catalyzes the inactivation of thrombin. An SIU of ACLM is therefore analogously defined as the amount that, in plasma containing 1 mumol of ATIII, will raise the (pseudo-)first-order breakdown constant of thrombin by 1 min-1. Of any given heparin preparation one can determine the specific HAM and ACLM activities in terms of SIU/mg. On the basis of the factor Xa and thrombin breakdown constants found in a plasma sample one can then determine the levels of HAM and ACLM. Preliminary experiments were carried out in plasma samples obtained after subcutaneous injection of unfractionated heparin (UFH) and of two types of low-molecular-weight heparin (LMWH). About three times more of UFH activity than of LMWH activity has to be injected to obtain the same levels of ACLM in the plasma. Only with the LMWHs significant amounts of BCLM are found, which rises higher and persists longer than the ACLM. We determined the course of thrombin generation in platelet-rich plasma (PRP) and in platelet-poor plasma (PPP), as well as in the PPP factor Xa generation curve and the course of prothrombin conversion. The observed inhibitions correlated much better with the levels of ACLM than with those of below critical chain length material. The difference between UFH and LMWHs can therefore not be explained in terms of antithrombin and anti-factor-Xa activity. The essential difference between UFH and LMWH appears in the feedback effect of thrombin in PRP, where thrombin generation is both inhibited and retarded by LMWH, while it is only retarded but hardly inhibited by UFH.     

Establishing a New Target Range for Unfractionated Heparin for Acute Coronary Syndromes

INTRODUCTION: The target activated partial thromboplastin time (aPTT) range of 1.5 to 2.5 times the control value or 45 to 75 seconds recommended by the ACC/AHA for patients receiving unfractionated heparin (UFH) for acute coronary syndromes (ACS) is vulnerable to variation in test reagents. Rather than use the standard target aPTT range, it has been recommended that each institution establish its own target aPTT range based upon anti-factor Xa heparin levels. As a quality assurance project, we evaluated our institution's therapeutic aPTT range by examining the correlation between aPTTs and anti-factor Xa heparin levels and established a new target aPTT range with a new thromboplastin reagent based upon the therapeutic anti-factor Xa heparin levels. METHODS: Sixty-two plasma samples from 26 consecutive patients receiving UFH for ACS were analyzed. Plasma aPTTs measured with a thromboplastin reagent and a new thromboplastin reagent and anti-factor Xa heparin levels were obtained on each plasma sample. Linear regression analysis was performed to establish a new target aPTT range from corresponding therapeutic anti-factor Xa heparin levels. RESULTS: Thirty-two percent of patients with our institution's target range aPTTs of 61 to 100 seconds had anti-factor Xa heparin levels below 0.35 to 0.7 U/mL while 68% of patients had therapeutic anti-factor Xa heparin levels (positive predictive value = 68%). When the same blood was tested with a new thromboplastin reagent lot, only 9% of patients with target range aPTTs had anti-factor Xa heparin levels below 0.35 U/mL while 91% of patients had therapeutic anti-factor Xa heparin levels (positive predictive value = 91%). The Pearson correlation coefficient ( r ) for the new thromboplastin reagent lot was 0.93. The target aPTT range established with the new thromboplastin reagent lot was 61 to 100 seconds. CONCLUSION: Monitoring aPTTs without standardizing the thromboplastin reagent may not adequately reflect therapeutic heparin levels. Despite apparently target aPTTs, patients treated with UFH may be under-anticoagulated. Our new anti-Xa-adjusted target aPTT range shows an increase in the positive predictive value of aPTTs. Large-scale clinical studies are needed to determine the optimal anti-factor Xa range for ACS patients treated with UFH, with further refinements if glycoprotein IIb/IIIa inhibitors are concomitantly used and to show a benefit in clinical outcomes for monitoring plasma heparin levels with anti-factor Xa heparin levels. Institutional standardization of the aPTT is necessary to ensure optimal patient care when changing thromboplastin reagents.     

The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.     

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin- catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.     

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4)

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.     

Treatment of heparin-associated thrombocytopenia and thrombosis with low molecular weight heparin (CY 216)

This report concerns 34 cases of heparin-associated thrombocytopenia in which standard heparin has been systematically replaced by LMW heparin CY 216 Choay. There were 21 women and 13 men, mean age, 69 years. Twenty-six of the 34 cases had thrombotic complications. All of the patients were treated by standard heparin of porcine mucosal origin, in most cases for prevention of deep vein thrombosis and pulmonary embolism. Twenty-six cases occurred along with orthopedic and traumatologic surgery (especially for total hip replacement, 15 cases). Four cases involved vascular surgery and three involved medical thrombotic disease. Standard heparin treatment was always replaced by LMW heparin CY 216. Initial doses were 0.30 ml three times daily, later increased to obtain global hypocoagulability. Surgical procedures (embolectomy and vena cava filter) were carried out when necessary. Five patients had thrombolytic treatment (urokinase) in conjunction with LMW heparin. The overall results were 31 recoveries, but with seven severe sequelae (three amputations and four hemiplegias) most often attributable to the first ischemic attack, and three deaths. For the last 16 patients, indirect platelet aggregation tests were performed (control platelet-rich plasma plus patient platelet-poor plasma plus LMW heparin): nine cases had negative tests, with nine recoveries and seven cases had positive tests, with five recoveries and two deaths.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of sulfation on antithrombin-thrombin/factor Xa interactions in semisynthetic low molecular weight heparins.

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.     

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

Summary. 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.     

Determination of low molecular weight heparin in clinical laboratory

The applicability was investigated of automated spectrophotometric heparin assays and three clotting assays for determination of two low molecular weight (LMW) heparin fractions: Org 10172 and DxN10 and two infractionated commercially available heparins. The relative activity of the two commercially available heparins was similar in the anti-Xa assay, in the anti-IIa assay and in 3 clotting assays. The LMW heparins showed markedly different relative activity in all 5 assays. The activities of those heparin preparations relative to the standard heparin were compared in the 5 assays, but standardization against a standard heparin preparation appeared impossible. Methods of heparin determination can be used to monitor treatment with a heparin preparation only if the same preparation is used as a reference substance.     

Laboratory monitoring of a low molecular weight heparin (enoxaparin) with a new clotting test (Heptest)

A new simple clotting test (Heptest) for low molecular weight heparins was compared to anti-Xa determination by an amidolytic assay in volunteers and in patients receiving standard calcium heparin or low molecular weight (LMW) heparin (Enoxaparin) by subcutaneous administration. The results obtained with both methods are in very good agreement. It seems that the Heptest, although influenced by various other clotting parameters, is nevertheless relatively specific for anti-Xa activity. Despite our positive first impression, we object to expressing the results in micrograms of heparin per milliliter or anti-Xa units. Until rigorous standardization is possible, we prefer to express the results as clotting times. Preliminary results warrant a more extensive study in order to assess the clinical relevance of Heptest in patients receiving unfractionated or LMW heparins.