Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.     

Modulation of fibrin clot formation by human serum amyloid P component (SAP) and heparin

Serum amyloid P-component (SAP) is a normal plasma constituent in man with a circulating concentration of approximately 40 micrograms/ml. Supraphysiological amounts of SAP (150-300 micrograms/ml) have been reported to affect coagulation. We have investigated this further by studying the effect of SAP upon clot times in both the absence and presence of heparin, a suggested ligand for SAP and itself a modulator of coagulation processes. In the absence of heparin, SAP (5-125 micrograms/ml) had no effect on clot times generated by Activated Thrombofax Reagent, brain thromboplastin, Russell's Viper Venom or thrombin when assessed in normal citrated plasma. However, in the presence of amounts of heparin that had only a minor effect upon clot times, SAP (5-40 micrograms/ml) greatly prolonged clot formation, with the thrombin time the most sensitive to SAP. This suggested that the primary effect of SAP was at this distal level of the coagulation pathway. Evaluation by radioimmunoassay revealed that supraphysiological concentrations of SAP (150-300 micrograms/ml) alone reduced by approximately 25% the release of fibrinopeptide A (FPA) from fibrinogen. In the presence of heparin, substantial synergism was observed with maximal reductions of approximately 70% in FPA production requiring only 25-50 micrograms/ml SAP. This inhibition correlated with increased thrombin clot time but was unrelated to any direct modulation in either the activities of anti-thrombin III or activated Factor XIII, and was independent of an alteration in the rate of fibrinolysis. Further, while SAP itself did not interfere with the process of spontaneous fibrin polymerization, in the presence of heparin a prolonged polymerization time (greater than 145%) was observed. We believe that these data reflect the primary mechanisms by which serum amyloid P component influences blood coagulation.     

Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C.

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.     

Revisiting antithrombin in health and disease,congenital deficiencies and genetic variants,and laboratory studies on α and β forms

Antithrombin [AT] is the main inhibitor for activated plasma coagulation serine esterases, inhibiting thrombin, Factors Xa and IXa, but also Factors XIIa, XIa, VIIa, kallicrein, and plasmin. Its activity is highly enhanced by heparin, through binding to the pentasaccharide sequences, for inhibition of all coagulation proteases, except thrombin, which inhibition requires its additional binding to the heparin polysaccharide chain. However, AT is the major inhibitor of thrombin in the blood circulation. Congenital or acquired deficiencies of AT expose affected patients to an increased risk of developing unprovoked and recurrent thrombo-embolic diseases. Antithrombin can be measured with various laboratory techniques, by either immunological or functional methods. Earlier, a radial immunodiffusion immunoassay allowed measurement of the protein antigenic content. Functional assays are mainly designed with Anti-Thrombin or Anti-Factor Xa chromogenic methods and are useful for detecting genetic molecular mutations with decreased inhibitory activity and contributed to study the conformational changes of antithrombin and its variants, which potentially regulate the activity of this serine protease inhibitor. These assays are not equivalent in terms of diagnosing protein abnormalities, associated with increased thrombotic incidence, and they have variable performance for reflecting impaired antithrombin binding capacity for heparin, reduced progressive inhibition of serine proteases, or accelerated switch rates to the latent and less active forms. A small proportion of AT (<10%) is present in blood in the β-form, with a lower oligosaccharide content, a lower Molecular Weight, a higher binding rate to endothelial glycosaminoglycans, and a higher anticoagulant activity, hence requiring specific laboratory methods for its measurement. The β-AT form is then of critical importance for controlling blood activation by tissue injury and preventing development of thrombo-embolic diseases. This article reviews the performance characteristics of the currently available assays, and their usefulness for monitoring the use of AT concentrates in intensive care units, disseminated intravascular coagulation or severe infections, to restore the anticoagulant protective effect of heparin by supplementing the requested AT concentration. The issues of automation, harmonization and standardization are also revisited and discussed.     

Argatroban as an alternative to heparin in extracorporeal membrane oxygenation circuits

We investigated the anticoagulant effects of argatroban, a direct thrombin inhibitor, versus heparin in extracorporeal membrane oxygenation (ECMO) circuits. Three sham circuits were prepared according to our hospital's standard practice and run for six hours simultaneously. Two circuits were anticoagulated with argatroban (one with heparin in the wet prime and one without). One circuit had heparin in the initial prime and was then anticoagulated with heparin. We measured thrombin generation (prothrombin fragment 1+2, D-dimer and thrombin-antithrombin complexes), activated clotting times (ACTs) and partial thromboplastin times (aPTTs), and monitored thrombus formation using thromboelastography. ACTs were >1000 s in each circuit throughout assessment. No clot initiation was detected by thromboelastography. Thrombin generation was decreased in circuits anticoagulated with argatroban versus heparin, despite aPTTs being less prolonged. These results suggest that argatroban may be more efficacious than heparin for anticoagulation in ECMO. Additional studies are warranted to further evaluate argatroban in this setting.     

Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder.

A woman, aged 68, with multiple myeloma (immunoglobulin[Ig]A kappa type) developed an anticoagulant with properties suggestive of heparin. The anticoagulant prolonged the thrombin time but not the reptilase time and was resistant to boiling, proteolytic enzyme digestion, and trichloracetic acid precipitation. The thrombin time was corrected by the addition (in vitro) of protamine sulfate or the addition of purified platelet Factor 4 (PF4) to the plasma. The anticoagulant was isolated by PF4-Sepharose affinity chromatography and analyzed in terms of its molecular weight, uronic acid, and amino acid composition. The proteoglycan isolated had a mol wt of 116,000 and appears to consist of two 38,000 dalton polysaccharide units interconnected by peptide material totaling 39,000 daltons. Electrophoretic analysis of the pronase digested peptidoglycan using the lithium acetate-agarose technique suggested the material was of the heparan sulfate type. The peptidoglycan had about one-tenth the specific activity of commercially available heparin on a weight basis. The isolated proteoglycan was indistinguishable from commercial heparin when analyzed in terms of its ability to act as a cofactor in the antithrombin III inhibition of thrombin.     

Heparin, thrombin and Factor Xa inhibitors

Direct and indirect coagulation inhibitors are used to inhibit the activity of the serine proteases of the coagulation system. Indirect inhibitors act via antithrombin and heparin cofactor II. The main representatives are heparins, lowmolecular-weight heparins, fondaparinux, idraparinux and danaparoid. They bind to antithrombin and potentiate the inactivation of factor Xa and other serine proteases. Direct thrombin inhibitors bind reversibly to thrombin without cofactor. Anticoagulants are determined by global and specific anticoagulant methods. New anticoagulants are developed such as oral factor Xa inhibitors, oral thrombin inhibitors, antibody against activated factor VII, recombinant tissue pathway inhibitor to improve inhibition of blood coagulation or to induce nonanticoagulant effects (e. g. activated protein C in septicaemia). New anticoagulant methods are developed to improve and specify the anticoagulant effect of anticoagulants in thromboembolic diseases.     

Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4.

Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate. We conclude that histidine-rich glycoprotein and platelet factor 4 can regulate the antithrombin activity of heparin cofactor II.     

Inhibition of activated protein C by platelets.

Activated protein C (APC), an anticoagulant that acts by inactivating Factors Va and VIIIa, is dependent on a suitable surface for its action. In this study we examined the ability of human platelets to provide this surface and support APC-mediated anticoagulant effects. The activity of APC was examined in three systems: the Factor Xa recalcification time of Al(OH)3 adsorbed plasma, studies of thrombin generation in recalcified plasma, and assessment of the rate of inactivation of purified Factor Va. In comparison with phospholipid, intact platelets required significantly greater concentrations of APC to achieve a similar degree of anticoagulation. When washed platelet membranes were substituted for intact platelets, adequate support of APC was observed and the anticoagulant effect was similar to that obtained with phospholipid. Platelet releasate obtained by stimulation of platelets with thrombin and epinephrine contained an inhibitor that interfered with the ability of phospholipid and washed platelet membranes to catalyze the anticoagulant effects of APC. A noncompetitive inhibition was suggested by Dixon plot analysis of the interaction between platelet releasate and APC. The activity of the platelet APC inhibitor was immediate and was not enhanced by heparin, distinguishing it from the circulating protein C inhibitor. The presence of this inhibitor in the platelet and its release with platelet stimulation emphasizes the procoagulant role of this cell.     

Inflammo-coagulatory response, extrinsic pathway thrombin generation and a new theory of activated clotting time interpretation

When blood is subjected to contact with foreign surfaces, as during cardiopulmonary bypass (CPB), the whole body inflammatory response is initiated, resulting in the expression of procoagulant molecules on the vascular endothelium and white blood cells. These surface bound procoagulants participate in the extrinsic coagulation pathway. It appears that the primary source of thrombin generation during CPB is due to extrinsic pathway activation. Thrombin not only converts fibrinogen to fibrin, it also acts as a proinflammatory agent resulting in a positive feedback loop or the inflammo-coagulatory response. Extrinsic pathway thrombin generation occurs as a membrane bound event. Membrane bound factors are resistant to heparin/ATIII inhibition. Therefore, the anticoagulant effect of heparin/ATIII is due to thrombin inhibition, not the inhibition of thrombin generation. Interpretation of the activated clotting time (ACT) must take into account the thrombin concentration [T]; this results in the coagulatory ratio, ACT is proportional to ([Hep -ATIII]/[T]). Considering this proportionality, it can be seen that the ACT cannot be used to quantitate heparin concentration. Changes in the ACT can reflect changes in [Hep - ATIII], changes in [T], or changes in both concentrations. Anti-inflammatory agents which suppress or inhibit the extrinsic pathway, such as aprotinin, result in decreased thrombin generation. As thrombin generation decreases, the ACT-heparin dose response curve is warped, resulting in a dose response curve resembling a PTT-heparin dose response curve. We can no longer assume that the disproportionate rise in the ACT relative to the [HEP - ATIII] when aprotinin is used as indicative of failure of the ACT to provide a credible indication of anticoagulation.     

A rationale for targeting antithrombotic therapy at the vessel wall: improved antithrombotic effect and decreased risk of bleeding

Intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) or vascular surgical procedures remains a significant problem despite current antithrombotic therapy. The use of the current antithrombotic drugs, namely heparin + chronic aspirin (ASA) +/- oral anticoagulants, is based upon the assumptions that: i) heparin blocks thrombin generation and/or accelerates thrombin inhibition by antithrombin III (ATIII); ii) aspirin acetylates platelet cyclooxygenase, thereby preventing thromboxane A2 (TxA2) synthesis; and iii) oral anticoagulants reduce the availability of vitamin K-dependent procoagulants, thereby reducing the risk of thrombus formation. Albeit beneficial, this approach has a number of shortcomings and limitations: i) when thrombin binds to an injured vessel wall, it becomes resistant to inhibition by heparin/ATIII; thus, surface-bound thrombin remains active, stimulating further thrombus formation, smooth muscle cell proliferation and subsequent hyperplasia; ii) while TxA2 inhibition reduces platelet reactivity, platelets are able to respond to multiple stimuli generated at the time of, or after, vessel wall injury; and iii) heparin, aspirin and the oral anticoagulants all render the patient hemostatically defective and at risk of bleeding. Recent studies suggest that alternate therapeutic approaches can inhibit thrombogenesis more effectively at the time of injury, thereby not only inhibiting hyperplasia more effectively than the currently used drugs, but also reducing (or eliminating) the need for long-term therapy. For example, we suggest that the heparin cofactor II (HCII) catalysts, dermatan sulfate and Intimatan, inhibit surface-bound thrombin more effectively than heparin/ATIII, thereby inhibiting intimal hyperplasia effectively. Their effects are achieved when the drug is given only at the time of injury; i.e. with no further antithrombotic therapy. Other studies indicate that injured vessel wall thrombogenicity can be reduced by pretreatment with Persantine (dipyridamole) or with certain fatty acid supplements which either increase vessel wall cAMP and/or 13HODE synthesis. These increases are associated with decreased vessel wall thrombogenicity, which, in turn, is associated with decreased intimal hyperplasia. Such results suggest that vessel wall repair is achieved more effectively by targeting antithrombotic drugs directly at the vessel wall thrombogenicity per se rather than indirectly by altering the circulating blood cells and systemic coagulant system.     

Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.

Thromboembolism is a prominent but poorly understood feature of eosinophilic, or Loeffler's endocarditis. Eosinophil (EO) specific granule proteins, in particular major basic protein (MBP), accumulate on endocardial surfaces in the course of this disease. We hypothesized that these unusually cationic proteins promote thrombosis by binding to the anionic endothelial protein thrombomodulin (TM) and impairing its anticoagulant activities. We find that MBP potently (IC50 of 1-2 microM) inhibits the capacity of endothelial cell surface TM to generate the natural anticoagulant activated protein C (APC). MBP also inhibits APC generation by purified soluble rabbit TM with an IC50 of 100 nM without altering its apparent Kd for thrombin or Km for protein C. This inhibition is reversed by polyanions such as chondroitin sulfate E and heparin. A TM polypeptide fragment comprising the extracellular domain that includes its naturally occurring anionic glycosaminoglycan (GAG) moiety (TMD-105) is strongly inhibited by MBP, whereas its counterpart lacking the GAG moiety (TMD-75) is not. MBP also curtails the capacity of TMD-105 but not TMD-75 to prolong the thrombin clotting time. Thus, EO cationic proteins potently inhibit anticoagulant activities of the glycosylated form of TM, thereby suggesting a potential mechanism for thromboembolism in hypereosinophilic heart disease.     

Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists

BACKGROUND: Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. OBJECTIVE: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. RESULTS: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. CONCLUSIONS: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.     

The dermatan sulfate-dependent anticoagulant pathway is mostly preserved in aneurysm and in severe atherosclerotic lesions while the heparan sulfate pathway is disrupted

BackgroundThe pathogenesis of abdominal aortic aneurysm is associated with changes of several components of arterial wall. Vascular glycosaminoglycans contribute to the non-thrombogenic activity of blood vessels. We investigated whether modifications of glycosaminoglycans in human abdominal aortic aneurysm affect their anticoagulant properties.MethodsGlycosaminoglycans were extracted from abdominal aortic aneurysms (n = 11) derived from reconstitution surgeries, human abdominal aortas (n = 9) from normal organ transplant donors and from preserved (n = 10) and atherosclerotic (n = 17) segments obtained from autopsy of an old patient. Glycosaminoglycan composition, concentration and anticoagulant activity were determined.ResultsGlycosaminoglycans extracted from aneurysms have a more potent anticoagulant activity than those from normal arteries of young adults, mostly due to a relative enrichment of dermatan sulfate, which potentiates heparin cofactor II inhibition of thrombin. Arterial segments of aged patient with severe atherosclerosis showed a glycosaminoglycan composition similar to aneurysms samples. Glycosaminoglycans extracted from these regions showed also a more potent heparin cofactor II-dependent anticoagulant activity than lesion-free areas due to the relative enrichment of dermatan sulfate.ConclusionThe anticoagulant activity from abdominal aortic aneurysms is preserved. No modifications particular to the aneurysms were dissociated from those observed in atherosclerosis.     

Impaired Anticoagulant Effect of Heparin in the Artificial Kidney: An Experimental Study

Bjo?rnson, J. &; Godal, H. C. Impaired Anticoagulant Effect of Heparin in the Artificial Kidney. An Experimental Study. Scand. J. clin. Lab. Invest. 36, 581–589, 1976.

Dialysis of blood and plasma was performed in vitro, in a ‘mini-Kiil’ dialyser as well as in dialysis bags. A marked shortening of the thrombin-clotting time was observed, indicating fall in heparin anticoagulant effect. The concentration of heparin, however, as measured by polybrene titration, was substantially less reduced. Fibrin formation, as evidenced by the ethanol gelation test, occurred more often in the dialysed than in the control plasma. In conclusion, the discrepancy between concentration and anticoagulant effect of heparin could be partly explained by influx from the dialysate of calcium, magnesium, and acetate ions. The fibrin-polymerizing effect of these ions was confirmed by a shortening of the clotting time with Reptilase, a proteolytic enzyme not influenced by thrombin inhibitors such as heparin. In addition, liberation of platelet factor 4 may be responsible for some reduction in antithrombin activity of heparin. No evidence of heparin being dialysed or adhering to the dialysis membrane was found.     

Studies of human serum amyloid P-component (sap) in relation to coagulation

Serum amyloid P-component (SAP) undergoes calcium-dependent binding to certain polysaccharides and polyanions. It has been claimed that it acts as a heparin antagonist and that plasma SAP levels are low in patients treated with vitamin K antagonist anti-coagulant drugs. However, in the present study depletion of SAP from plasma had no effect on subsequent coagulation, indicating that it is not a necessary procoagulant. SAP levels in male patients receiving warfarin were the same as in normal controls; in a group of female patients on warfarin, SAP levels were slightly higher than normal, probably in response to the underlying condition for which they were anticoagulated. The SAP from warfarin-treated individuals was indistinguishable from normal SAP antigenically, electrophoretically and in its calcium-dependent ligand binding. Isolated SAP did not inhibit the anti-coagulant activity of heparin, on the contrary, addition of supraphysiological amounts of isolated SAP to citrated plasma or to mixtures of fibrinogen and thrombin inhibited clotting, possibly by interfering with fibrin polymerisation. C-reactive protein (CRP), the classical acute phase reactant, which is closely related to SAP, did interfere with the neutralisation of factor Xa by heparin. This effect was produced by concentrations of CRP within the range commonly seen in disease states, suggesting that it may be the counterpart of an in vivo role for CRP in the acute phase response.     

Production of thrombi on intact endothelium by use of antiheparin agents in vivo

It had been suggested that antithrombin activity on the surface of intact endothelial cells may play a role in inhibiting platelet adhesion and thrombus formation. The antithrombin activity may be due to thrombomodulin or to activation of antithrombin III by glycosaminoglycans or thrombomodulin, or possibly a combination of these. This inhibitory activity has been shown to be affected by such antiheparin agents as protamine, hexadimethrine bromide (Polybrene; Aldrich Chemical Co., Milwaukee, Wis.) and platelet factor 4, as well as by such enzymes as heparinase and heparitinase. We have used a hamster cheek pouch preparation to observe thrombus formation in vivo in a normal vascular flow, to determine whether the production of thrombi by thrombin can be enhanced by antiheparin agents. After intra-arterial injection or topical application of protamine or hexadimethrine bromide, platelet adhesion and thrombus formation on intact arteriolar endothelium was produced by a dose of thrombin, which when injected alone had no effect. No thrombi were found in venules or capillaries. Injection of heparin before or after the antiheparin agents necessitated a larger dose to enhance the action of thrombin. On electron microscopy the thrombi were found to consist primarily of platelets adherent to an intact endothelium. The possible clinical implications of these observations are discussed.     

Increased concentrations of heparin cofactor II in diabetic patients, and possible effects on thrombin inhibition assay of antithrombin III

We compared concentrations of antithrombin III (AT-III) in plasma, as determined by an immunological method and by a functional thrombin inhibition method, in the presence of heparin in 160 blood samples from Type I diabetics. Although the correlation was highly significant (P less than 0.001) between the results obtained by the two methods, our data demonstrated that results by the thrombin inhibition assay, 121 (SD 15)%, expressed as percentages of the results for a normal plasma pool, were significantly (P less than 0.001) higher than by the immunoreactive method, 104 (SD 15)%, indicating an overestimation of functionally active AT-III. Concentrations of functionally active AT-III determined by a factor Xa inhibition assay, 105 (SD 13)%, were in the same range as immunoreactive AT-III. Addition of IgG antiserum to normal pooled plasma quenched only about 90% of the AT-III activity determined by the thrombin inhibition assay, but all of the AT-III activity determined by a factor Xa inhibition assay. These results demonstrate that the factor Xa inhibition assay is more specific for the determination of AT-III than the thrombin inhibition assay. We suggest that the high concentrations of heparin cofactor II, 117 (SD 17)%, might have caused an overestimation of AT III in this group of patients with diabetes Type I, and should not be overlooked in other clinical situations.     

From heparin to heparins: toward new therapeutic perspectives?

Heparin is mainly known for its anticoagulant action, but today other biological effects are investigated. With the low molecular weight heparin fractions (LMWH), more homogenous, a more detailed study of the mechanism of action of heparins can be made. The anticoagulant action of heparin is mainly antithrombin III (AT III) dependent and the binding site of AT III on the heparin molecule has been recently identified. LMWH have a lower anticoagulant (anti-IIa) activity, and a relatively higher anti-Xa activity (ratio anti-Xa/anti-IIa = 5 to 10 for LMWH and 1 for standard heparin). The antithrombotic action of heparins is not strictly correlated to their anticoagulant activity. Other mechanisms of action, such as interactions with vascular endothelial cells and the fibrinolytic system may contribute to the antithrombotic action of heparins. New therapeutical possibilities are currently under investigation. Inhibition of vascular smooth muscle cells growth by heparin suggest a possible control of the atherosclerotic process by heparin. Moreover, heparin and its derivatives might be involved in the regulation of the cellular growth process.     

Antithrombotic properties of a direct thrombin inhibitor with a prolonged half-life and AT-mediated factor Xa inhibitory activity

Summary.  Rebound thrombin generation after successful thrombolysis might be related to (i) too short-term anticoagulant therapy and to (ii) the inability of heparin derivatives to inhibit clot-bound thrombin. To meet these shortcomings, a compound was synthesized, which consists of a pentasaccharide conjugated to a direct thrombin inhibitor. This compound (Org 42675) has a 10 times longer half-life compared with the original half-life of the direct thrombin inhibitor, while the thrombin inhibitory activity is maintained. An extra advantage of this product is the inhibitory activity on thrombin generation via antithrombin III (AT)-mediated factor (F)Xa inhibition. Org 42675 inhibited in vitro clot-bound thrombin with similar activity to the direct thrombin inhibitor argatroban. In experimental models in rats, Org 42675 showed on a molar base similar antithrombotic activity to unfractionated heparin, was more active than argatroban and was more active than fondaparinux sodium (AT-mediated FXa inhibitor) in arterial thrombosis. Finally, Org 42675 was far more active than the three reference compounds in an experimental thrombolysis model in rabbits. These properties of Org 42675, with its FXa and (clot-bound) thrombin inhibitory activity in combination with its long half-life, make this compound a powerful drug that is likely to be effective in the prevention of re-occlusion after successful thrombolysis in man.