Nonfatty acid-modulated variations in drug binding due to heparin.

Changes in free fatty acids (FFAs) do not always correlate with variations in drug binding. To dissociate heparin effects on FFAs and drug binding, six healthy fasting subjects received 50 units (USP) IV heparin (Harris L932) with and without protamine (3.2 mg IV). Protamine completely suppressed heparin-induced rises in FFAs and warfarin free fraction (W alpha), but diazepam free fraction (D alpha) increased (P less than 0.005). In vitro, increasing concentrations of heparin added to serum increased D alpha (P less than 0.0005) and W alpha (P less than 0.005) without changing FFAs. In eight subjects given 50 units IV heparin (Harris L014), FFAs (P less than 0.001) and propranolol free fraction (P alpha) rose (P less than 0.01), but variations in FFAs and P alpha did not correlate (r = 0.18). When two different heparin lots (Harris L014 and Organon LA39) were tested in vivo. Harris L014 heparin increased FFAs (P less than 0.005) and P alpha (P less than 0.0005), but variations in FFAs and P alpha correlated poorly (r = 0.43, P less than 0.05). In contrast, the Organon LA39 heparin did not change FFAs, but did increase P alpha (P less than 0.0005); variations in FFAs and P alpha did not correlate (r = 0.22). These results indicate that heparin-induced variations in drug binding are not exclusively related to changes in FFAs.

Variability in heparin effect on serum drug binding.

Heparinized saline was given to seven men and one woman, aged 21 to 42 yr, after a 14-hr fasting period and 2 hr after breakfast; blood was collected in nonoheparinized tubes. Diazepam (D alpha) and warfarin (W alpha) free fractions were determined in serum by equilibrium dialysis to which radiolabeled drug was added. After 50 U heparin (Harris LO14) intravenously, the maximum effect on D alpha, W alpha, and free fatty acids (FFA) developed in 5 min and lasted 20 to 30 min. D alpha rose and W alpha fell (p < 0.01) at 5 min. Cumulative doses of heparin increased FFAs (F4,16 = 18.29, p < 0.0005). D alpha rises (r = 0.73, p < 0.001) and W alpha falls (r = -0.74, p < 0.001) correlated with changes in FFAs. D alpha rises and W alpha falls were greater postprandially than in the fasted state (p < 0.01). Five subjects were randomly assigned up to 400 U intravenously of each of two different heparin lots (Harris LO14, and Organon LA39.) The FFA rises (reflecting heparin lipolytic activity, F1,32 = 179.62, p < 0.0005), D alpha rises (F1,32 = 34.22, p < 0.0005), and the W alpha falls (F1,32 = 33.20, p < 0.0005) by heparin Harris LO14 were greater than those by heparin Organon LA39. Although small doses of heparin, such as those in heparin locks, can affect drug binding, the extent and variability of the effect depends on the biologic activity of the heparin, and varies with manufacturer and lot, exact time of sampling, and eating.

A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next?

Heparin is a sulfated glycosaminoglycan isolated from animal organs that has been used clinically as an antithrombotic agent since the 1940s. In the early 1980s it was discovered that a unique pentasaccharide domain in some heparin chains activates antithrombin III (AT-III), a serine protease inhibitor that blocks thrombin and factor Xa in the coagulation cascade. Sanofi-Synthélabo and Organon developed a synthetic analogue of this pentasaccharide. The resulting antithrombotic drug arixtra, which went on the market in the USA and Europe in 2002, shows superior antithrombotic activity and brings about AT-III-mediated activity against factor Xa exclusively. Structure-based design has subsequently led to analogues with longer-lasting activity, such as idraparinux, as well as novel conjugates and long oligosaccharides with specific anti-Xa and antithrombin activities. The new drug candidates are more selective in their mode of action than heparin and less likely to induce thrombocytopenia.

An attempt to standardize APTT reagents used to monitor heparin therapy.

Citrated samples from 100 patients on i.v. heparin and 20 normal patients were tested with three batches each of three activated partial thromboplastin time (APTT) reagents: Thrombosil I (Ortho); Automated APTT (Organon Teknika) and Actin FSL (Baxter). The ratio of APTT over the geometric mean normal APTT for each heparinized sample was calculated. One batch of reagent arbitrarily chosen as a reference gave the ratios APTRREF (y). The remaining reagents to be standardized against the reference system gave the ratios APTRTEST (x). The best correlation between systems was given by log vs log x. Standard curves were prepared from the APTT ratios of the 20 normal patients and 65 of the heparinized samples. On plotting log APTRTEST vs log APTRREF the y intercept was close to zero so x was expressed in terms of y using; log x = HSI. log y, where HSI (Heparin Sensitivity Index) = slope. The APTRTEST results of the remaining 35 heparinized samples were transformed using; APTRTRANS = (APTRTEST)HSI.APTRTRANS was then compared to APTRREF to determine whether the transformation brought the results closer to the reference. We conclude that although some improvement was found by using the transform, it was not possible to mathematically relate APTT results due to a high degree of variation between results using different reagents. A standard APTT reagent for the monitoring of heparin therapy is recommended. A separate APTT reagent may be required for the screening of factor deficiencies and lupus anticoagulants.

Comparison of the effect of a conventional heparin and a low molecular weight heparinoid on platelet function.

In a study comparing the in vitro effects of heparin and a low molecular weight heparinoid (Organon 10172) on aggregation of platelets from normal subjects, we have demonstrated that whereas heparin markedly enhances platelet aggregation induced by other aggregators and inhibits the anti-aggregatory effect of epoprostenol (prostacyclin, PGI2), heparinoid does not produce such effects. The use of heparinoid may thus have a significant advantage over that of heparin in situations where enhanced platelet aggregation is the main factor leading to thrombosis or where heparin treatment is followed by thrombocytopaenia.

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.