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

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

Appearance of heparin antithrombin-active chains in vivo after injection of commercial heparin and in anaphylaxis

Recently it has been demonstrated that only one-third of commercial heparin binds with antithrombin III to enhance antithrombin activity, and that heparin is rapidly taken up by the RES. Commercial heparin was given to rabbits, dogs and men by subcutaneous and intravenous injection. Plasma samples were deproteinized with phenol, urine samples precipitated with benzidine, and the separated heparin measured for both antithrombin and metachromatic (dye-binding) activity compared to a reference heparin. Urinary excretion as heparin and uroheparin accounted for approximately 5% of the injected heparin. The extraction of heparin added to blood $\text{in}$$\text{vitro}$ gave identical values in metachromatic and antithrombin assays, as did heparin from blood samples immediately after intravenous injection. However, later blood samples after injection gave much higher values in the test for antithrombic activity than in the metachromatic test. Subcutaneous injection of heparin subjected to acid inactivation in vitro, reducing its antithrombin activity, likewise resulted in heparin appearing $\text{in}$$\text{vivo}$ with higher antithrombin activity. Endogenous heparin released to the circulation in canine anaphylaxis also gave much higher values in anti-thrombin assays. It is concluded that the difference in values is due to the conversion of antithrombin-inactive heparin chains to antithrombin-active heparin chains.     

Anticoagulant and antithrombin effects of intimatan, a heparin cofactor II agonist

Surface-bound thrombin, which is resistant to inhibition by heparin/antithrombin III (/AT), plays a key role in vessel wall disease. In contrast, surface-bound thrombin is not resistant to inhibition by heparin cofactor II (HCII) and its acceleration of its inhibitory effect by dermatan sulfate. However, the potential use of dermatan sulfate to prevent thrombus formation in vivo is limited by its low specific activity, which in turn, necessitates excessively high doses when given on a gravimetric basis. Recently, a novel HCII agonist, Intimatan, has been synthesized by site-specific sulphation of highly purified dermatan sulfate comprising primarily of L-iduronic acid-4-O-sulphated N-acetyl-D-galactosamine, yielding a 4, 6-O-disulphate compound on the galactopyranose ring with a lower molecular weight, higher solubility, and specific activity than its parent, dermatan sulfate. In this study, we compared the abilities of Intimatan with its parent compound, dermatan sulfate, and with heparin to affect coagulation and to inhibit surface-bound thrombin both in vitro and in vivo, to determine if Intimatan demonstrates a better potential than either other compound in preventing thrombus formation in vivo. Intimatan prolonged the activated partial thromboplastin time (APTT) more effectively than either dermatan sulfate or heparin at comparable antithrombin concentrations. This activity was attributed to the more selective action of Intimatan against surface-bound thrombin in vitro. Intimatan also inhibited thrombin bound to an injured vessel wall surface in vivo more effectively than heparin, i.e., when measured in injured carotid arteries of rabbits injected with Intimatan or with heparin at the time of injury. We conclude that Intimatan effectively inhibits surface-bound thrombin, thereby exhibiting better anticoagulant and antithrombin properties than heparin and dermatan sulfate.     

Pronounced effects of the combination of a new thromboxane antagonist (GR32191) and heparin on bleeding time in man.

Potential pharmacokinetic and pharmacodynamic interactions between two oral doses of GR32191 (40 and 80 mg), a new thromboxane antagonist, and heparin (5,000 IU bolus + 1,000 IU/h for 3 h) were studied in eighteen healthy male volunteers using two separate double-blind, randomised, placebo-controlled, cross-over studies. Mean (range) bleeding time values were 8.4 min (7.5-9.7) during heparin/placebo, 12.1 min (9.2-18.6) (GR32191/placebo) and 16.3 min (11.5-21.4) (GR32191/heparin) in the 40 mg study, while these values were 8.7 min (5.5-15.5), 16.0 min (9.3 - greater than 36.0) and 23.8 min (10.7 - greater than 36.0), respectively in the 80 mg study. Compared to screening values, the combination of 80 mg of GR32191 and heparin had a greater effect on the bleeding time than the sum of the prolongations after the separate treatments (p = 0.05). In the 40 mg study this was not the case. Pharmacokinetics of heparin (as assessed by plasma anti-Xa and antithrombin activity) and GR32191 were unaltered during co-administration of the two drugs. GR32191 did not influence the effects of heparin on APTT. Heparin slightly diminished the inhibition of collagen induced platelet aggregation by 80 mg of GR32191 and the U-46619 (thromboxane A2-mimetic) induced platelet aggregation remained unchanged. Overall fibrinolytic activity (as evaluated by the fibrin plate test) was similar during all three treatments in the study with 80 mg. The combination of 80 mg of GR32191 and heparin caused a prolongation of the bleeding time which was more than expected on the basis of their individual effects.     

Heparin and fibrinolysis--comparison of subcutaneous administration of unfractionated and low molecular weight heparin

The effects on the fibrinolytic system after a single s.c. bolus injection (at 9 a.m.) of either 5000 IU conventional heparin or 5000 anti-Xa U of a fractionated low molecular weight heparin (Fragmin, KabiVitrum, Sweden) were investigated in 9 healthy volunteers. The effects were compared to those of an injection of normal saline in 6 volunteers. Samples for biochemical analyses were taken regularily during 6 hours after drug or placebo administration. In the coagulation system the following parameters were measured: Activated partial thromboplastin time (APTT), anti-Xa activity, thrombin time and fibrinogen. The fibrinolytic system was monitored by analysing: plasminogen, alpha 2-antiplasmin, fibrin(ogen) degradation products (FDP), euglobulin clot lysis time (ECLT), tissue plasminogen activator (t-PA) activity, t-PA antigen and plasminogen activator inhibitor (PAI) activity. Injection of the 2 drugs was followed by elevations in APTT and anti-Xa activity, and were more pronounced for Fragmin than heparin. The fibrinolytic system exhibited a diurnal variation with decreasing PAI activity and increasing t-PA activity during the day. Volunteers receiving normal saline (placebo) showed a similar pattern. The results were unrelated to heparin. It is concluded from this study that neither heparin nor Fragmin had any significant effect on the fibrinolytic parameters when measured after a single s.c. bolus injection since the observed variations were within the diurnal range.     

Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.     

Profibrinolytic activity of the direct thrombin inhibitor melagatran and unfractionated heparin in platelet-poor and platelet-rich clots

Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.     

Effect of standard heparin and a low molecular weight heparin on thrombolytic and fibrinolytic activity of single-chain urokinase plasminogen activator in vitro

The effect of unfractioned heparin (UH) and low molecular weight heparin (LMWH) (Kabi 2165 - Fragmin) on in vitro scu-PA thrombolytic and fibrinogenolytic activity was investigated. Thrombolytic activity was evaluated by following lysis of radiolabeled plasma clot immersed in plasma in presence of scu-PA alone or with either form of heparin. A 200 IU/ml scu-PA concentration produced clot lysis within 7 hr. UH or LMWH led to a slightly faster clot lysis which was statistically significant only at the 2nd and 3rd hour. No significant difference could be evidenced between UH and LMWH effect. During clot lysis, plasmin, generated within the clot led to a gradual transformation of scu-PA to tcu-PA, specially after a 4-hr incubation. Appearance of tcu-PA activity in the plasma surrounding the clot was significantly inhibited by either form of heparin. This finding contrasts with results observed in purified systems and suggests the presence of heparin-dependent plasma factor(s) inhibiting tcu-PA formation or its activity. Possible candidates might be anti-thrombin III and PAI-3. No fibrinogen breakdown was observed when plasma was incubated for 7 hr at 37 degrees C in presence of scu-PA alone (200 IU/ml) or with either form of heparin. However, in presence of a plasma clot, an important fibrinogen breakdown was observed during clot lysis reflecting the action of plasmin and/or tcu-PA generated within the clot, in the surrounding plasma. Fibrinogenolysis was less pronounced in the presence of both heparin preparations possibly as a consequence of the reduction in the tcu-PA level. These results underline the importance of plasma factors in the interaction of heparin with plasminogen activators such as scu-PA.     

Pharmacokinetics of UH and LMWH are similar with respect to antithrombin activity

BACKGROUND: The ability to administer low molecular weight heparins (LMWH) subcutaneously without laboratory monitoring contributes to their popularity for the treatment of thrombotic disorders. Subcutaneous unfractionated heparin, although less expensive, is deemed to require routine laboratory monitoring on the basis of more variability in drug effect compared to LMWH. However, the more predictable pharmacokinetic profiles of low molecular weight heparins are largely based on anti-Xa activity, while antithrombin activity may be at least as important to their mechanisms of action. METHODS: We performed a clinical pharmacokinetic trial to compare the variability in peak antithrombin effect between subcutaneous unfractionated heparin and various LMWHs, all given in recommended weight-adjusted treatment doses. Sixty-one patients enrolled in a warfarin clinic were randomized to receive one of four different weight-adjusted subcutaneous heparin doses: unfractionated heparin, 250 units/kg (n=15); tinzaparin, 175 units/kg (n=15); dalteparin, 200 units/kg (n=15); or enoxaparin, 1 mg/kg (n=16). The areas under the curves of antithrombin levels during the first 3 h after administration were determined for each patient, and the coefficients of variation (CV) and 95% confidence intervals of the AUCs were compared among the treatment groups. RESULTS: There was no statistically significant difference in the coefficients of variation of antithrombin effect between unfractionated heparin (52.8, 95% CI: 32.6-72.9) and enoxaparin (56.5, 95% CI: 35.7-77.4) or dalteparin (43.5, 95% CI 25.4-61.6). Tinzaparin had statistically significant decrease in coefficients of variation (21.6, 95% CI: 12.2-30.9) relative to unfractionated heparin, dalteparin and enoxaparin. CONCLUSIONS: LMWHs, as a class of drugs, are no more predictable in antithrombin effect after subcutaneous injection than unfractionated heparin. There were considerable differences among LMWHs in the observed variability of antithrombin effects, with tinzaparin being somewhat more predictable than the other drugs tested.     

Fibrinolytic and anticoagulant activity after a single subcutaneous administration of a low dose of heparin or a low molecular weight heparin-dihydroergotamine combination

The anticoagulant and potential profibrinolytic effect of a combination of low molecular weight heparin with dihydroergotamine (LMWH-DHE) and of unfractionated heparin was studied in eight healthy volunteers. Each volunteer received a subcutaneous injection of either LMWH-DHE (1,500 U anti-Xa of LMWH + 0.5 mg DHE), unfractionated heparin (5,000 IU) or of placebo (saline) between 7 and 8 h in the morning on three different occasions. Anti-Xa activity, and fibrinolytic activity measured by the euglobulin clot lysis time (ECLT) and by the fibrin plate assay were determined before and at different times after administration of the three substances. Anti-Xa activity in plasma reached a maximum four hours after injection of both LMWH-DHE and unfractionated heparin. LMWH-DHE showed a better bioavailability when compared to unfractionated heparin; the anti-Xa activity peak was two and a half fold higher after LMWH-DHE despite injection of a three fold lower dose of anti-Xa units. The half-life of anti-Xa activity was approximately 4 hours for LMWH-DHE but only 90 min for unfractionated heparin. The fibrinolytic activity measured by ECLT as well as by fibrin plate assay, showed a significant increase during the day reaching a peak 8-12 h after injection regardless of the product administered (including the placebo). The profile of the diurnal fibrinolytic activity curve was identical for all three substances. The increase in fibrinolytic activity, observed after administration of LMWH-DHE or unfractionated heparin, was therefore not due to these drugs but reflected the circadian physiological fluctuation of fibrinolysis.     

Elimination of intravenously administered radiolabelled antithrombin III and heparin in humans

Antithrombin III was purified from normal plasma by DEAE-Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-antithrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S-radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin - antithrombin III in equilibrium heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.     

Heparin compromises streptokinase-induced arterial patency in rabbits

INTRODUCTION: Little is known with regard to efficacy of heparin as an adjunct to fibrinolytics under conditions of severe vascular damage. In this study, we compared the effects of unfractionated heparin (UH), low-molecular weight heparin (LMWH), and recombinant desulfohirudin (HIR) in combination with streptokinase (SK) in such settings. MATERIALS AND METHODS: We used an established rabbit model, in which thrombosis, critical stenosis, and vascular wall damage were introduced to a segment of the abdominal aorta and the effects of the respective therapies were assessed by time to patency (TTP in minutes), cumulative patency (CP (%)), lysis of original clot (CL (%)), and net clot accretion (NCA (%)). Treatments were administered over 90 min at the following doses: SK: 33,000 U/kg, UH: 125-250 U/kg, LMWH: 1.25-2.5 mg/kg, HIR: 0.25-0.55 mg/kg. RESULTS: Unexpectedly, UH and LMWH had a paradoxical and detrimental effect on SK-mediated recanalization as measured by both TTP and CP. Thus, administration of SK vs. SK+UH or SK+LMWH resulted in TTP values of 43+/-8 min vs. 70+/-11 min (p<0.05) and 67+/-12 min. (p<0.08), respectively. For CP, the corresponding values were 21+/-7%, 0.5+/-0.3% and 9+/-8%. This delay in vessel recanalization occurred despite excessive systemic anticoagulation (activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) ratios >6 and >34, respectively). Of interest, both heparinoids completely inhibited SK-induced fibrinogen consumption (FC). In contrast, recombinant desulfohirudin (HIR) shortened SK-induced TTP (4.97+/-0.81 min) without preserving fibrinogen. CONCLUSIONS: Our findings suggest that caution needs to be exercised, when using the combination of SK and heparinoids for the treatment of arterial thrombosis under conditions of severe vascular damage and stenosis.     

Benefit/risk profile of high-dose antithrombin in patients with severe sepsis treated with and without concomitant heparin

A randomised, prospective, placebo-controlled phase III multicentre clinical trial (KyberSept) has been performed to test the efficacy of high-dose antithrombin therapy in patients with severe sepsis. Concomitant low-dose heparin has been routinely given in two thirds of patients for deep vein thrombosis prophylaxis. This study analyses heparin - antithrombin interactions in terms of long-term mortality, adverse events, and thromboembolic events. From a total of 2,314 patients with severe sepsis (placebo: n = 1,157; antithrombin: n = 1,157) 1,616 patients (placebo: 811, antithrombin: 805) received heparin concomitantly with study drug (antithrombin 30,000 IU) over four days, whereas 698 patients (346 and 352, respectively) did not. In patients with no concomitant heparin, 28-day mortality was lower with antithrombin than with placebo (37.8% vs. 43.6%; absolute reduction: 5.8%; risk ratio: 0.860 [0.725-1.019]), which increased until day-90 (44.9% vs. 52.5%; absolute reduction: 7.6%; risk ratio: 0.851 [0.735-0.987]). In patients with concomitant heparin, no effect of antithrombin on mortality was seen (28-day mortality: 39.4% vs. 36.6%; absolute increase: 2.8%; risk ratio: 1.08 [0.96-1.22]). Frequency of use of concomitant heparin increased during conduct of the study. Increased bleeding incidences were reported with antithrombin plus concomitant heparin as compared to antithrombin alone. Rates of thromboembolic events were similar when antithrombin was given with or without concomitant heparin. In the treatment of severe sepsis, high-dose antithrombin may sufficiently protect against development of venous thromboembolism when no concomitant heparin is given. Combined administration of the two increases bleeding risk and probably abolishes efficacy of antithrombin.     

Pharmacokinetics of fragmin. A comparative study in the rabbit of its high and low affinity forms for antithrombin

A tritium-labelled low molecular weight heparin fragment with an average molecular weight of 4000-6000 (Fragmin), was fractionated into its high and low affinity forms for antithrombin. The fractions obtained were injected into rabbits, and the plasma half-life (t1/2) volume of distribution (Vd), area under the curve (AUC), total body clearance (TCl) and renal clearance (Clr) were determined. When followed by radioactivity, both the high and low affinity forms of Fragmin as well as Fragmin itself were eliminated from plasma in a biexponential manner. Values for AUC were very low compared to those expected from the given dose. This effect was most pronounced for low affinity-Fragmin demonstrating a significantly higher extravascular distribution of molecules lacking affinity for antithrombin. From radio activity data, it was also demonstrated that the fraction of dose that was eliminated from plasma via non-renal (cellular clearance) mechanisms was higher for heparin (95 per cent) than for Fragmin (77 per cent) after a dose of 100 micrograms/kg. This demonstrates that cellular clearance is of less importance in the plasma elimination of low molecular weight heparin fragment, an effect that may explain their longer plasma half-lifes despite the fact that they are more readily and faster excreated into the urine.     

Human protein C induces anticoagulation and increased fibrinolytic activity in the cat

The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the cat was examined. Cats received bolus i.v. injections of PCa of 3 micrograms/mL (n = 7), 10 micrograms/mL (n = 2) or 16 micrograms/mL (n = 1) of calculated total blood volume. Control cats (n = 7) received either unactivated Protein C (10 micrograms/mL) or the activation vehicle containing heparin and antithrombin in tris-saline buffer. Citrated blood samples were drawn prior to infusion and 5, 10, 20, 40, 60, 120 and 180 minutes post infusion. PCa caused an immediate, dose dependent increase in anticoagulation as measured by APTT which began to normalize within 20 minutes. PCa also increased fibrinolytic activity measured by euglobulin clot lysis time and an 125I whole blood clot lysis assay. However, the profibrinolytic effect of PCa did not parallel the anticoagulant effect. In all treated cats, maximum fibrinolytic activity did not occur until 40-120 minutes after infusion. Control cats demonstrated no significant change in coagulation or fibrinolytic activity. Our results demonstrate that infusion of activated human Protein C can induce anticoagulation and fibrinolytic activity in the cat.     

Antithrombin III "Northwick Park": a variant antithrombin with normal affinity for heparin but reduced heparin cofactor activity

Further studies have been carried out in a previously reported family with congenital antithrombin III (AT III) deficiency due to an abnormal variant of AT III (AT III Northwick Park). The variant has been identified in five members of the family, three of whom had a history of venous thrombosis. Inheritance followed an autosomal dominant pattern. The affected family members have reduced levels of antithrombin heparin cofactor (41-67%) and progressive antithrombin activity (44-62%) but normal levels of immunoreactive AT III (91-162%). Two dimensional immunoelectrophoresis (2 DIE) of AT III in the absence of heparin revealed an abnormal fast-moving peak in addition to the normal peak but 2 DIE in the presence of heparin appeared normal. Further studies confirmed that the abnormal AT III binds completely to heparin but has no heparin cofactor or progressive antithrombin activity. These results would be consistent with a mutation affecting the binding site for thrombin.     

The inactivation of thrombin and plasmin by antithrombin III in the presence of sepharose-heparin.

The inhibition of thrombin and plasmin by antithrombin III was studied in the presence of heparin conjugated to Sepharose. When either enzyme was adsorbed to the heparin conjugate in quantities insufficient to occupy all the affinity sites, subsequent passage of antithrombin III through the column invariably produced complete inhibition as measured by the response to the chromogenic substrate S2160. However, when the loading sequence was reversed (i.e. adsorbing non-saturating quantities of antithrombin III before the enzyme), antithrombin III only insignificantly inhibited either enzyme over a 20-hour period. A second load of antithrombin III following the thrombin load resulted in complete inactivation. When thrombin was rendered incapable of binding to heparin by cyclohexanedione treatment and reacted with Sepharose-heparin to which antithrombin III was adsorbed, the protease inhibitor exhibited ‘progressive’ antithrombin activity. These studies may indicate that thrombin and plasmin possess higher affinities for heparin than for the antithrombin III-heparin complex. Furthermore antithrombin III reacts more readily with the enzyme-heparin complex than with heparin. If the relative affinities of heparin, thrombin and antithrombin III in plasma are similar to those observed under these affinity chromatographic conditions, then the present data are consistent with the view that heparin promotes the inactivation of thrombin and plasmin by augmenting their reaction with antithrombin III. An allosteric effect of heparin on thrombin is apparent from the 50% increase in esterase activity of thrombin on α-N-benzoyl-L-arginine ethyl ester which is observed in the presence of optimal concentrations of heparin.     

Antithrombin BM from human plasma: an antithrombin binding moderately to heparin

A human antithrombin was purified app. 60 fold from Cohn fraction IV, to give a single band of about 70.000 molecular weight in polyacrylamide gel electrophoresis. Compared to the similar antithrombin III, this glycoprotein binds only moderately to porcine heparin (hence its name Antithrombin BM), thus requiring higher heparin concentration for full thrombin inhibitor function, and lower ionic strength for elution from a heparin sepharose column. In these respects it resembles "heparin cofactor A", which is, however, characterized by a substantially larger molecular weight. From AT III, AT BM further differs in its absolute dependency on the presence of heparin(oids) for antithrombin activity, in its more pronounced inhibitory specificity largely restricted to thrombin, and in the absence of substantial immunological crossreaction with antibody to AT III. Based on comparative measurements of antithrombin activity in the presence of different amounts of heparin, up to 40% of the antithrombin activity present in human blood may be attributed to AT BM. The in vivo role of this new inhibitor remains to be elucidated.     

Identification of two distinct heparin cofactors in human plasma. II. Inhibition of thrombin and activated factor X

Heparin alone has two direct effects on the thrombin clotting time. First, there is an immediate prolongation of the clotting time, which can be fully reversed upon neutralization of the heparin by polybrene or protamine. Second, there is a slow, time-dependent increase in the clotting time, representing an irreversible decrease in the concentration of active thrombin. Increasing the heparin concentration above 10^?2 Units/ml leads to an increase in the rate of thrombin inactivation in the presence of cofactor A. This lost thrombin cannot be recovered by neutralization of the heparin. In the absence of heparin, cofactor A has only slight progressive antithrombin activity. Cofactor B by itself has marked progressive antithrombin activity, the rate of which is further enhanced by the addition of increasing concentrations of heparin above 10^?2 Units/ml. However, heparin diminishes the ultimate thrombin capacity of cofactor B. Cofactor B also inactivated activated Factor X (Factor Xa). The effect is greatly accelerated in the presence of heparin. Cofactor A has no effect on Factor Xa, even in the presence of heparin.     

Factor Xa inactivation by a heparinized hydrogel

Incubating Factor Xa and plasma in the presence of "beads" of a heparinized hydrogel (heparin-PVA) resulted in a loss of Factor Xa activity as measured by clotting time or chromogenic substrate assays. This loss in activity was attributed to the activity of the heparin immobilized to the polyvinyl alcohol (PVA) hydrogel. The immobilized heparin retained an apparent activity approximately 1% that of a similar mass of heparin in solution. The loss in apparent activity was in part attributed to diffusion effects which limited the accessibility of the enzyme and inhibitor to the heparin immobilized in the interior of the gel "beads". The chromogenic substrate activity of adsorbed Factor Xa was also measured and was found to decrease after exposure to antithrombin III to similar extents regardless of the sequence of exposure. This suggests that Factor Xa and antithrombin III have similar affinities for this immobilized heparin, unlike the situation for thrombin (Thromb-Res., 20, 543-554, 1980).