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.     

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.     

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.     

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。     

A sensitive colorimetric assay for thrombin, prothrombin and antithrombin III in human plasma using a new synthetic substrate

Benzoyl-L-leucyl-L-alanyl-L-arginine-alpha-naphthylester (Bz-Leu-Ala-Arg-NE) was synthesized as a new substrate for use in the assay of thrombin. In the assay alpha-naphthol released by the enzyme reaction was measured colorimetrically. With Bz-Leu-Ala-Arg-NE as substrate, the minimum detectable concentration of human thrombin was 0.0025 U. This assay using Bz-Leu-Ala-Arg-NE is a highly sensitive method for detecting prothrombin, thrombin and antithrombin III in human plasma. Prothrombin could be determined with 0.2 microliter of human plasma using Echis carinatus venom (ECV) as activator. Antithrombin III activity could be determined with 2 microliter of human plasma using human thrombin and heparin as cofactor. A zymogram of human prothrombin was prepared with Bz-Leu-Ala-Arg-NE as substrate. The preparation gave one band (pI 4.9) on polyacrylamide disc gel isoelectrophoresis.     

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.     

Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to the endothelium.

We have examined the role of heparinlike molecules in the regulation of coagulation by perfusing rat hindquarters with purified human thrombin and with its plasma inhibitor, antithrombin. Our data indicate that contact of the hemostatic components with the endothelium enhances the rate of thrombin-antithrombin complex formation by as much as 19-fold over the uncatalyzed rate of enzyme-inhibitor interaction. Heparinlike molecules are responsible for the antithrombin accelerating activity. The amount of thrombin-antithrombin complex generated within the hindlimb preparation after pretreatment of the vasculature with purified Flavobacterium heparinase or with addition of platelet Factor IV to the hemostatic components, was equal to the uncatalyzed levels. These heparinlike molecules appear to be tightly bound to the luminal surface of the endothelium, since they could not be detected within the physiologic buffer that was perfused through the animal. The above mucopolysaccharides function in a manner similar to commercial heparin, since modification of antithrombin at a site critical for heparin-dependent acceleration of the protease inhibitor resulted in a level of interaction product identical to the uncatalyzed amount. Finally, addition of diisofluorophosphate-thrombin to the enzyme perfusion stream reduced the amount of thrombin-antithrombin complex formed in the animal by 30-40%, which suggested that thrombin bound to the endothelium as well as enzyme free in solution are accessible to antithrombin that has interacted with heparinlike molecules present on the endothelium.     

Abnormal antithrombin III with defective serine protease binding (antithrombin III "Denver")

A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and antithrombin activity is described. Plasma levels were: AT-III ag, 0.92-0.96 U/ml; AT-III heparin cofactor activity, 0.54-0.62 U/ml; progressive antithrombin activity index, 0.13-0.18; anti-Xa activity, 0.50-0.56 U/ml. Plasma crossed immunoelectrophoresis (CIE) patterns performed with and without added heparin were normal, but serum CIE revealed a decreased complex peak. Purification of the patient's plasma AT-III by heparin-sepharose affinity chromatography showed a normal protein recovery and elution profile, but the purified AT-III fraction showed only 50% of the normal progressive thrombin neutralization and anti-Xa activity. When thrombin-antithrombin (TAT) complexes were formed by incubating with excess thrombin, SDS-polyacrylamide gel electrophoresis (PAGE) analysis revealed that half the patient AT-III formed TAT complexes while the remainder migrated as free AT-III. All the control AT-III formed TAT complexes. The patient's nonreacting AT-III (AT-III "Denver"), isolated by affinity chromatography, showed CIE and SDS-PAGE migration patterns characteristic of normal AT-III but failed to bind thrombin or Xa. Calculations from turnover studies in one patient and normal subjects with autologous 131I-AT-III suggested that AT-III "Denver" is removed from the plasma slightly more rapidly than normal. These studies indicate that the patients' variant AT-III molecule was characterized by normal heparin interaction but defective binding and inhibition of thrombin and Xa. These characteristics allow isolation of the nonreactive variant molecule by heparin-sepharose affinity chromatography.     

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin

Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.     

Heparin cofactor II activity in plasma during pregnancy and oral contraceptive use

Inhibition of thrombin by heparin is mediated by at least two plasma proteins, antithrombin III, and heparin cofactor II. The plasma titer of heparin cofactor II was significantly elevated in both pregnant women and users of oral contraceptives.     

高半胱氨酸对血管内皮细胞,血小板聚集和肝素辅助因子活性？…

目的 揭示高半胱氨酸（Ｈｃｙ）致血栓形成的机制。方法 （１）免疫荧光分析务管内皮细胞凝血酶调节蛋白（ＴＭ）抗原分布;（２）发色基质法测定肝素辅助因子Ⅱ（ＨＣⅡ）活性。结果 （１）Ｈｃｙ可下调血管内皮细胞表面ＴＭ的表达,且呈浓度依赖性;（２）Ｈｃｙ对血浆ＡＴ－Ⅲ和ＨＣⅡ的抗凝血酶活性无明显影响,在纯化的ＡＴ－Ⅲ的抗凝血酶作用也无影响。结论 Ｈｃｙ的致血栓效应与其对内皮细胞ＴＭ表达调节有关,且独立于血     

Human alpha2-macroglobulin and its antitrypsic and antithrombin activities in serum and plasma.

alpha2-Macroglobulin level, trypsin protein esterase and progressive antithrombin activities were measured in normal and nephrotic sera and plasma. Trypsin protein esterase activity was proportional to the alpha2-macroglobulin concentration in serum and plasma from both normal and nephrotic patients. The results were different, however, with progressive antithrombin activity: in normal plasma, antithrombin III is the main thrombin inhibitor, then alpha2-macroglobulin and alpha1-antitrypsin, whereas in nephrotic syndrome patients, alpha2-macroglobulin is the main thrombin inhibitor.     

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.     

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.     

IMPURITY OF THROMBIN PREPARATIONS FOR CLINICAL USE

Recent advances in the treatment of upper gastro-intestinal bleeding has led to the use of thrombin preparations in this condition. In the present investigation, the purity of thrombin preparations from one American pharmaceutical company and two Japanese pharmaceutical companies was examined using immunological and biological methods. An SDS-PAGE study revealed that each preparation contained various proteins other than thrombin. An immunological study revealed that each of the thrombin preparations contained proteins which were of at least two types. The thrombin preparations exhibited various biological activities other than thrombin activity, and their activities were widely different. Preparation A showed the strongest activity among the three, in terms of amidolytic activity, γ-γ dimer production rate, fibrin production rate, release of fibrinopeptide A, and anti-stretch activity. However, preparation A revealed the strongest binding activity to antithrombin III. Preparation B had the least contamination while preparation C showed the highest activity in terms of fibrinolytic and platelet-aggregating activity. Thus, each thrombin preparation had a greater or lesser degree of impurity, and it would appear that purer preparations are required for local administration in the treatment of upper gastro-intestinal bleeding.     

Formation of factor Va by atherosclerotic rabbit aorta mediates factor Xa-catalyzed prothrombin activation.

Vascular cell procoagulant activity may be important in the pathogenesis of atherosclerosis. In previous studies, we described the ability of the atherogenic metabolite homocysteine to activate endothelial cell Factor V, a key coagulation cofactor for thrombin generation. The present study was designed to investigate Factor V activity and Factor Xa-catalyzed prothrombin activation by control and atherosclerotic aorta from normal and hypercholesterolemic rabbits. Factor Xa generated ninefold more thrombin on atherosclerotic aortic segments than on control segments. Atherosclerotic segments activated 125I-prothrombin with Factor Xa in the presence of the thrombin inhibitor dansyl arginine-4-ethylpiperidine amide and cleaved 125I-Factor V. This suggests that increases in vessel-wall Factor V activity and Factor Xa-catalyzed prothrombin activation result from activation of vessel-wall Factor V. 125I-Factor Va peptides generated by atherosclerotic aorta were very similar in molecular weight to those generated by homocysteine-treated cells. When vascular endothelium was mechanically removed by brushing, atherosclerotic vessels still generated four- to fivefold more thrombin than control vessels. These data and results from immunocytochemical studies suggest that Factor V in atherosclerotic vessels is associated with both endothelium and other cells of the lesion. In contrast, Factor V in control vessels is associated primarily with endothelium. The increases in Factor V activity and thrombin formation in the blood vessel wall of hypercholesterolemic rabbits may contribute to the development of atherosclerosis and its complications.     

A kinetic micromethod of determining the activity of antithrombin III

A method for measuring the blood plasma antithrombin III (AT III) activity is suggested, based on measurement of the rate of inactivation of added standard volume (0.05 ml) of plasma thrombin AT III. The thrombin residual activity in the mixture is detectable over the course of fibrinogen tests. Curves are plotted, reflecting the rate of inactivation of thrombin AT III in the tested and reference plasma samples; by modeling and comparing these curves the values of the examined sample AT III activity are obtained, with the effects of fibrinogen and fast antithrombins various levels eliminated.     

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.     

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.     

Neutralization of the anticoagulant effects of glycosaminoglycans by serum amyloid P component: comparison with other plasma and platelet proteins.

Serum amyloid P protein (SAP) is a heparin-binding protein that is found in blood and connective tissues including some types of vascular basement membrane. In this article we present evidence that SAP is capable of blocking the anticoagulant effects of glycosaminoglycans. SAP neutralized the catalytic effect of heparin on the thrombin-antithrombin III reaction more effectively than vitronectin, histidine-rich glycoprotein, fibronectin, and high-molecular-weight kininogen and almost as effectively as platelet factor 4. SAP also blocked the effects of heparin and dermatan sulfate on the inhibition of thrombin by heparin cofactor II. We found evidence for the formation of a high-affinity 1:1 complex between SAP and heparin and for inhibition of binding of both thrombin and antithrombin III to heparin-Sepharose by SAP. We conclude that SAP may account for much of the heparin-neutralizing capacity of plasma under some conditions and that basement-membrane-bound SAP may modulate extravascular coagulation by blocking the anticoagulant effects of basement membrane glycosaminoglycans.