刺参酸性粘多糖介导肝素辅因子Ⅱ对凝血酶活性的抑制

目的：进一步澄清刺参酸性粘多糖（Ｓｊａｍｐ）的抗凝血酶机制。方法：用国产Ｓｊａｍｐ作为激动剂，在正常混合血浆体系、纯化的肝素辅因子Ⅱ（ＨＣⅡ）体系以及纯化的抗凝血酶Ⅲ（ＡＴ－Ⅲ）体系研究Ｓｊａｍｐ的抗凝血酶作用机制。结果：Ｓｊａｍｐ对凝血酶的抑制主要呈ＨＣⅡ依赖性，当存在ＨＣⅡ时，Ｓｊａｍｐ抗凝血酶作用的二级速率常数Ｋ２＝１．５６×１０７ｍ－１·ｍｉｎ－１，其抑制速率常数是ＡＴ－Ⅲ的４．６倍。结论：在抗凝血酶作用方面，Ｓｊａｍｐ的效率（Ｋ２值）及机制（ＨＣⅡ依赖性）与硫酸皮肤素类似。     

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 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.     

Inhibitory activities for thrombin and factor Xa of whale heparin: comparative studies on two preparations from different species

Whale intestinal heparin preparations from Balaenoptera physalus L. (Bp) and Balaenoptera borealis L. (Bb) were fractionated by affinity chromatography on a column of antithrombin III (AT)-Sepharose 4B. The yields of high-affinity fractions for AT (HA) from Bp and Bb were 86.8 and 13.3%, respectively, in total. The inhibitory activities for thrombin of Bp and Bb and their HA and those for Factor Xa of the latter were compared in the presence of AT. The results indicated that the inhibitory activities for thrombin of Bp and its HA were higher than those of Bb and its HA, respectively. No noticeable difference in the inhibitory activity for Factor Xa was, however, observed between HA from Bp and Bb. The present observations confirm our previous assumption that the presence of the thrombin-binding regions in addition to the AT-binding regions in heparin molecule are essential for the manifestation of high inhibitory activity for thrombin in the presence of AT.     

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.     

Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin

Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. OBJECTIVE: Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.     

Effect of protamine sulfate on antithrombin III activity

When protamine sulfate was added to heparinized plasma in vitro for neutralization of heparin, the activities on both thrombin and Xa known as heparin cofactor in antithrombin action were completely abolished. However, progressive activities on thrombin and Xa both recovered within 30 minutes after protamine sulfate addition. When equivalent heparin was again added, heparin cofactor activity was immediately restored. Based on the fact that protamine sulfate did not show any direct action on the antithrombin III molecule, the presence of AT III with progressive activity was considered to play an important role in the rebound phenomenon of heparin after heparin neutralization with protamine sulfate.     

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.     

Identification in vitro of an endothelial cell surface cofactor for antithrombin III. Parallel studies with isolated perfused rat hearts and microcarrier cultures of bovine endothelium.

Two in vitro systems were used to identify an antithrombin III cofactor activity on vascular endothelium. Langendorff rat heart preparations or columns packed with endothelium cultured on microcarrier beads were perfused with mixtures of purified thrombin and antithrombin III. With each preparation, accelerated inhibition of thrombin by antithrombin III occurred during passage over endothelium. Platelet factor 4, protamine sulfate and diisopropylphosphoryl thrombin, all antagonists of the antithrombin III cofactor activity of heparin, significantly reduced the capacity of the preparation to inhibit thrombin. It is concluded that a substance with the functional properties of a stationary phase cofactor for antithrombin III is present on the microvascular endothelium and there catalyzes the inactivation of circulating free thrombin.     

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 in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Clinical grade heparin is a very heterogeneous mucopolysaccharide, containing molecules with Mr ranging from 6,000 to 30,000 that have either a high affinity or a low affinity for antithrombin III (AT). In this study, the antithrombotic properties of intact high-affinity heparin (Mr = 15,000) and of two heparin fragments (h16, a 16-monosaccharide fragment, with Mr = 4,300, and h12, a 12-monosaccharide fragment, with Mr = 3,200) and of their functional covalent stoichiometric complexes with human AT were compared in a venous thrombosis stasis model in rabbits. Thrombosis was induced by injection of glass-activated human plasma and measured in a segment of the jugular vein that was isolated between two vascular clamps for 10 min. Injections of 55 micrograms/kg resulted in a clear antithrombotic effect for intact heparin, but not for the two fragments. Equivalent amounts (carbohydrate moiety) of covalent complexes of heparin or of both heparin fragments with human AT resulted in an antithrombotic effect lasting for 45-60 min. Injection of 110 micrograms/kg of heparin and of the heparin fragments yielded an antithrombotic effect, lasting 45-60 min; the corresponding amounts of covalent complexes caused an anti-thrombotic effect for 60-120 min. The free and conjugated fragments produced equal antithrombotic effects at equal plasma levels of anti-Factor Xa activity, but the specific antithrombotic activities of free and complexed intact heparin, on a molar basis, were 10-20-fold greater than those of the free and complexed heparin fragments. The plasma half-life of the covalent complexes of the heparin fragments with AT is, however, 10 times longer than that of the complex between intact heparin and AT and 30 times longer than that of free intact heparin. Covalent complexes between AT and heparin fragments could, therefore, be useful to maintain more stable levels of antithrombotic activity in plasma.     

Purification and characterization of hereditary abnormal antithrombin III with impaired thrombin binding

Investigations of a family predisposed to recurrent venous thromboses disclosed a hereditary antithrombin III deficiency. The reactive antithrombin III concentration in plasma was reduced approximately 50%, and the antigen concentration of the inhibitor was normal. Antithrombin III from two members of this family was purified by dextran sulfate precipitation, affinity chromatography on heparin-Sepharose, and ion-exchange chromatography on DEAE-Sephadex A-50. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis showed that only approximately half of the purified antithrombin III was capable of forming a complex with thrombin. This corroborated the finding that approximately twice as much purified antithrombin III from these patients compared with antithrombin III from normal humans was needed for titration of a given amount of thrombin. The nonreactive as well as the reactive population of antithrombin III bound heparin with the same affinity as normal antithrombin III. This was shown by crossed immunoelectrophoresis using heparin in the first dimension, by the elution pattern during salt gradient elution of antithrombin III from heparin-Sepharose, and by heparin enhancement of intrinsic fluorescence. Kinetic studies in the absence and in the presence of heparin indicated that the fraction of antithrombin III that could inactivate thrombin was functionally normal. The affected family members appeared to be heterozygotes with two autosomal codominant alleles that encode a normal and an abnormal antithrombin III protein, respectively.     

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.     

Development and characterization of an automated assay of effective heparin activity in plasma

We describe a fully automated assay for determining effective heparin activity in plasma, based on heparin-catalyzed inhibition of Factor Xa (EC 3.4.21.6) by antithrombin III (AT III). Residual Factor Xa is determined kinetically by the Du Pont aca discrete clinical analyzer with a chromogenic substrate and is inversely related to heparin activity. Because the test plasma is the sole source of AT III, the assay result is dependent on AT III activity and reflects effective rather than total heparin activity. The assay range is 20-1200 USP units/L, and the assay shows equivalent sensitivity to standard and low-molecular-mass heparins. Within-run reproducibility (CV) is 1.6% at 390 units/L. There was no interference from common blood components or drugs. Results agreed well with those by the Coatest heparin kit (Kabi) adapted to the Cobas-Bio analyzer (r = 0.85, n = 122).     

Involvement of heparin cofactor II in chymotrypsin neutralization and in the pancreatic proteinase—antiproteinase interaction during acute pancreatitis in man

Heparin cofactor II is a proteinase inhibitor which inhibits both chymotrypsin and thrombin, and displays great similarities with antithrombin III, the main inhibitor of thrombin in human plasma. Since acute pancreatitis is known to be associated with modification of the proteinase-antiproteinase equilibrium, we studied heparin cofactor II and antithrombin III as well as other biochemical and haematological parameters in 10 patients experiencing attacks of acute pancreatitis. Heparin cofactor II activity decreased during the first week of illness, while its antigen concentration remained subnormal. This discrepancy between antigen concentration and activity which persisted during the first week of illness was due both to complex formation of heparin cofactor II with its target proteinases and to partial proteolysis of the inhibitor. Heparin cofactor II was shown to form a complex with chymotrypsin in the plasma of such patients. Antithrombin III levels remained unchanged throughout the study, with no discrepancy between its activity and antigen concentration. No modification of haemostasis was shown either, except for a rise in the fibrinogen level during the first days of illness. It is concluded that, unlike antithrombin III, heparin cofactor II is involved in the proteinase-inhibitor equilibrium in patients with acute pancreatitis, and that heparin cofactor II might react as an inhibitor of pancreatic proteinases rather than an inhibitor of thrombin.     

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin‐independent mechanism

Summary. Background: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin‐independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity. Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC₅₀s) of low molecular weight heparin (LMWH), super‐sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX‐dependent and FIX‐independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock‐depleted or antithrombin/FIX‐depleted plasma supplemented with recombinant FIX. Results: UFH and fondaparinux demonstrated similar potency under FIX‐dependent and FIX‐independent conditions, whereas LMWH (2.9‐fold) and ssLMWH (5.1‐fold) demonstrated increased potency with limiting TF. UFH (62‐fold) and fondaparinux (42‐fold) demonstrated markedly increased EC₅₀ values in antithrombin‐depleted plasma, whereas LMWH (9.4‐fold) and ssLMWH (two‐fold) were less affected, with an EC₅₀ within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin‐depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin‐binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin‐independent interaction with the FIXa heparin‐binding exosite.     

Lipolytic and anticoagulant activities of a low molecular weight fragment of heparin

Low molecular weight heparin (LMWH) and standard heparin were given intravenously to six healthy subjects receiving a continuous infusion of Intralipid. After infusion, antifactor Xa, antithrombin II and coagulation activity (Normotest) were the same for both heparins. Activated partial thromboplastin time increased significantly, but the increase was much higher after standard heparin (+473%) than after LMWH (+48%). The increase in lipoprotein lipase activity was less pronounced after LMWH infusion. This resulted in a smaller decrease in Intralipid-triglyceride concentration and a smaller increase in both plasma FFA concentration and Intralipid fractional removal rate compared to standard heparin. This study shows that the plasma lipolytic potential of LMWH is weaker than that of standard heparin when given in doses with equipotent anticoagulation. LMWH may therefore be preferable to standard heparin as an antithrombotic agent in clinical situations where a high plasma lipolytic activity may be disadvantageous.     

A method of determining heparin in blood plasma based on its antithrombin activity]

The suggested method for measuring blood plasma heparin is based on heparin ability to enhance antithrombin activity of antithrombin III (AT-III), the major Xa and thrombin inhibitor. The method consists in measurement of blood plasma AT-III activity in the presence and absence of protamine sulfate that destroys the heparin--AT-III complex. Heparin content in U/ml is determined from the difference in the activities of heparin--AT-III complex and AT-III proper activity represented on the calibration curve. The method is sufficiently sensitive, it permits registration of heparin concentrations in a wide band (from 0.01 U/ml to 0.75 U/ml of plasma).     

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis

Summary.  Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin–protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and α₂-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.     

Identification of a congenital dysthrombin, thrombin Quick.

A dysprothrombin designated prothrombin Quick, is isolated from the plasma of an individual with < 2% of normal functional prothrombin activity and 34% of the normal prothrombin level by immunologic assay. With Factor Xa or taipan snake venom as activators, a fragmentation pattern identical to that of normal prothrombin is observed on gel electrophoresis in dodecylsulfate. This evidence combined with the observed barium citrate adsorption of prothrombin Quick and the low activity suggests that the defect in prothrombin Quick is in the thrombin portion of the molecule. Thrombin Quick is isolated and comigrates with thrombin on dodecyl sulfate gel electrophoresis, either reduced or nonreduced. The activity of thrombin Quick on several biological substrates of thrombin is investigated. Relative to normal thrombin, thrombin Quick is 1/200 as active on fibrinogen and 1/20-1/50 as effective in activating Factors V and VIII and aggregating platelets. A complex with antithrombin III is detected by dodecyl sulfate gel electrophoresis. Further investigation with the active site titrant, dansylanginine-N-(3-ethyl-1,5-pentanediyl)amide showed that the thrombin Quick preparation has the same affinity for the titrant as thrombin, but apparently only 40% active sites per mole protein are titrable.