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Heparin Cofactor Ii HEPARIN COFACTOR II 1549 1550 HEPARIN COFACTOR II ASSAY : IMPORTANCE AND CONTROL OF HEPARIN COFACTOR II: A SIMPLE ASSAY 1'1ETHOD: H. Vinazzer and THE NON-CONTAMINATION BY HEPARIN OF DERMATAN SULFATE. U.Pangraz. Blood Coagulation Laboratory,Linz,Austr1a J. Tapon-Bretaudiere, A.M. Fischer, C. Millien and A. Bros. Laboratoire d'Hematologie, Facult~ Necker, Paris, A photometric assay method for heparin cofactor II (HC II) is de­ France. scribed. In a first step antithrombin III (AT III) in plasma is blocked by an anti human AT III immunoglobuline from goats. After A chromogenic substrate method for the determination dilution of this plasma with Tris buffer pH 8.4 containing 3 IU/ml of heparin cofactor II (HC II) in plasma, based on its ~eparin and addition of thrombin the remaining thrombin activity anti Fe IIa activity, has been developped in our labo­ 1s measured by use of the chromogenic substrate S-2238 Kabi. The ratory, using dermatan sulfate (D.S.) as activator. We following preliminary experiments were carried out: Variation of observed that D.S. from porcine skin, which was claimed the amount of anti-AT III added to plasma resulted in complete not to be contaminated by heparin, was sometimes conta­ inactivation of 1.25 units AT III by 1.0 ml of the inhibitor. minated, depending upon the batch used. Moreover, a Incubation of 1 ml anti AT III·with 1 ml purified AT III ( 1 U/ml) classical treatment of D.S. by nitrous acid, that spe­ or with 1 ml normal plasma completely abolished AT III activity cifically degrades heparin-like glycosaminoglycans, is within 60 sec. Incubation of the reaction mixture with thrombin not always sufficient and in some cases, needs to be re­ resulted in maximum inactivation after 180 sec. This is in contr­ peated. It is thus important to check the absence of ast to AT III activated by heparin which immediately inactivates heparin contamination for the validity of the HC II thrombin. Anti-Xa activity after depletion of AT III was assayed assay. Indeed, we have shown that for an amount of con­ in a similar way by addition of factor Xa to the reaction mixture taminating heparin as small as 5x1o·S IU per ~g of D.S., and measuring the remaining Xa activity by the substrate S-2222. the HC II level of a normal plasma was increased by50%. In these tests no anti Xa-activity was found after AT III deplet­ Such a low contamination is undetected by routine tests ion. From these experiments there was assumed that the antithromb­ (thrombin time, APTT, heparin activity assay). Thus, we in activity measured under the followinq conditions was due to propose two useful methods for this purpose : (i) an the action of HC II: - anti-Fc Xa heparin cofactor activity assay on normal Plasma (50 ~1) was mixed with anti AT III (50 ~1) and was incub­ plasma, using D.S. as activator ; HC II being devoid of ated for 60 sec. Tris buffer with heparin pH 8.4 (900 ~1) was any anti-Fc Xa activity, if a Fe Xa inactivation is ob­ added. From this mixture 200 ~1 was pipetted into a cuvette at served, it is due to AT III potentiation by the conta­ 37oc followed by 200 ~1 thrombin ( 2 IU/ml). After an incubation minating heparin. (ii) an HC II assay using a normal time of 180 sec 200 ~1 S-2238 ( 2 mmol/1) was added and the diff­ plasma and an AT III depleted plasma ; in the case of erence in OD/min was determined at 405 nm. A calibration curve D.S. contamination, a higher level of HC II is obtained was made by series of dilutions of normal AT III depleted plasma with normal plasma, reflecting the AT III potentiation from 20 healthy individuals. The following preliminary results by heparin. For example, with 2.5x1o-5 IU of heparin of<·HC II activity as a percentage were obtained: healthy individuals (n=20) 100 ~ 9.2% per ~g"of D.S. : (i) an anti-Fc Xa activity of 50% was found, decreasing to 0% after decontamination, (ii) the liver cirrhosis (n=12) 64 ~ 14.1% nephrotic syndrome (n= 3) 94 ~ 8.8% HC II. level which was 125% in normal plasma, compared DVT (n=17) 89 ~ 10.6% to 100% in AT III depleted plasma was identical (100%) DIG (n= 9) 56 ~ 16.8% in both plasmas after decontamination. These results demonstrate the validity of the two methods and the usefulness of a control of each batch of D.S. used in HC II assay, even after nitrous acid treatment of the glycosaminoglycan. 1551 1552 KINETICS OF HEPARIN CO-FACTOR II (HCII) :THROMBIN AND EFFECT OF THE BUFFER COMPOSITION AND THE PRESENCE OF ORG 10172 ANTITHROMBIN Ill (ATIII):THROMBIN INTERACTION. MOLECULAR WEIGHT AND SOME GLYCOSAMINOGLYCANS ON THE INHIBITION OF ALPHA-THROMBIN DEPENDENCY. V.E. Ellis, M.F. Scully and V.V. Kakkar. BY HEPARIN COFACTOR II. Thrombosis Research Unit, King's College School of Medicine & I.M.A. Verhamme and G.W.K. van Dedem+ Dentistry, Denmark Hill, London SES 8RX, UK. Lab. of General Biochemistry and Physical Pharmacy,State Univ. of Gent,Belgium, and +Biochemical Research Lab., Diosynth BV ,Oss, The The influence of increasing concentrations of heparin of Nether lands. different molecular mass (Mr) has been compared in potentiation Heparin cofactor II (HCII) inhibits alpha-thrombin (IIa) with for­ of the rate of HCII: thrombin interaction and of ATIII: thrombin mation of a 1 :1 complex.The inhibition is accelerated by heparin interaction under pseudo first orde~ conditions. Unfractionated and dermatan sulfate (DS).The present study describes the pH and and fractionated heparin showed a concentration dependent ionic strength dependence of the second order inactivation con­ ascending and descending limb of stimulation of the rate which stant k2nd for the simple IIa-HCII interaction and for the inter­ was closely similar for both inhibitors. Unfractionated heparin action catalysed by heparin, dermatan sulfate and the antithrom­ and fractions of 16.5 KDa or less showed a peak acceleration of botic Org 10172 containing low molecular DS. In all cases we found the rate of interaction of thrombin with both inhibitors at a bell-shaped pH profile for k2nd with characteristic maxima and 0.3xlo-6M heparin although the observed maximum rate at this pK values. Org 10172 accelerates the HCII mediated inhibition of peak decreased with fall in Mr. For both inhibitors two high Mr II a though not to the same extent as heparin and DS. Substantial This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. fractions (22KDa and 32KDa) showed peak stimulation at a lower narrowing of the pH profile occurs under influence of glycosami­ heparin concentration (0.3xlo-7M) and 1.5 to 2 fold greater noglycans,the alkaline limb of the profiles being displaced to­ increase in rate than that observed with unfractionated wards a more acidic pH. The ionic strength is critical for the heparin. Under these conditions it could be calculated that the determination of k2nd. A nonlinear decrease is observed with in­ potency of a 32KDa fraction was l200iu/mg with respect to UF creasing ionic strength, due to the screening effect of the ions heparin (l50iu/mg). Three further pools were prepared and ATIII in solution. ca++ ions do not accelerate the inactivation process ,high affinity fraction prepared by chromatography. Acceleration in absence of GAG, in contrast to the findings for the IIa-ATIII of rate of interaction was measured according to concentration interaction. The pseudo-first order model for IIa inactivation is and inverse plots gave values for apparent Kd amd maximal rate. only valid for : a) the uncatalysed reaction irrespective of Apparent Kd (nM) Maximal rate k' ionic strength, b) the inactivation in buffers of intermediate (nM) (min-1) ionic strength and in the presence of low GAG concentration and Heparin pool (KDa) HCII ATIII HCII ATIII c) the inactivation in media of high ionic strength irrespective 27 2.7 1.2 2.2 2.22 of GAG concentration. In all other reaction conditions the inac­ 13 16.0 4.2 1.33 1.66 tivation rate substantially deviates from a pseudo-first order 7. 7 33.0 8.4 0.36 1.25 plot.Suicide substrates are known to exhibit a comparable beha­ These results suggest that differences in the profiles of vior.However this model appeared inadequate for describing our stimulation by high Mr fractions to those of lower Mr are experiments. An appropriate model for these aberrant inactivation related to higher binding affinities for the inhibitor profiles is not evident.Deviation from pseudo-first order kine­ permitting maximal binding of heparin before the descending part tics occurs more readily with heparin than with dermatan sulfate of the slope due to saturation of thrombin (according to the and is probably related with their difference in charge density. template hypothesis). Although close similarity was found between heparin stimulation of HCII and AT! II, potentiation of HCII inhibitory activity differed in that it was reversed by lower ionic strength and was not reversed by a heparin I pentasaccharide with high affinity for ATIII. 422 .
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