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Comparative effects of enoxaparin and unfractionated heparin in healthy volunteers on prothrombin consumption in whole blood during coagulation, and release of tissue factor pathway inhibitor
THROMBOSIS RESEARCH 89; 443-452,1993 0049-3848/93 $8.00 + .OOPrinted in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.
COMPARATIVE EFFECTS OF ENOXAPARIN AND UNFRACTIONATED HEPARIN IN HEALTHY VOLUNTEERS ON PROTHROMBIN CONSUMPTION IN WHOLE BLOOD
DURING
COAGULATION, PATHWAY
AND RELEASE INHIBITOR
OF TISSUE
FACTOR
L. Bara*, M.F. Bloch*, D. Zitoun”, M. &mama**, F. Collignon***, A. Frydman ***, A. Uzan***, J. Bouthier***. “Laboratoire de Thrombose Expkimentale, Universitk Pierre et Marie Curie, Paris, France,** Laboratoire Central d’Hkmatologie, Hbtel-Dieu, Paris, France, *** RhGnePoulenc Rorer, Recherche et DCveloppement, Antony, France. (Received 10.7.1992; accepted in revised form 23.12.1992 by Editor D.A. Lane)
ABSTRACT In a randomized crossover study twelve healthy male volunteers (23.5 + of 4.8 years, 73.0 + 6.4 kg, 180.8 f 5.7 cm) received one subcutaneous injection of either enoxaparin (EN) at 40 mg or 1 mg kg-l, or unfractionated heparin (UH) at 5,000 IU at one week intervals. Area under curves (AUC) of Anti-Xa and Anti-IIa activities correlated with EN dose. The relative effectiveness of EN versus UH 5,000 U as assessed by AUC ratio (EN/UH) was 7 and 15 for Anti-Xa activity, 1.3 and 3.1 for Anti-IIa activity after SC injection of EN 40 mg (4,000 Anti-Xa IU and 1,200 Anti-IIa’U) and 1 mg kg-l (7,300 + 640 Anti-Xa 1U and 2,190 + 290 Anti-IIa IU) respectively. In volunteers receiving EN, a dose dependent inhibition of thrombin generation rate in platelet depleted plasma (PDP), measured with a new and simple chromogenic thrombin generation assay, was observed when compared with baseline values. Similarly, intrinsic prothrombin activation in whole blood, evidenced by measuring residual factor II in serum 2 hours after clotting (prothrombin consumption test : PC), was inhibited in a dose dependent manner. In UH treated volunteers, although the inhibition of thrombin generation rate in PDP was similar to that observed with EN 40 mg, prothrombin consumption in whole blood was not significantly modified. Tissue factor pathway inhibitor (TFPI) activity release was increased similarly for UH and EN 40 ( 1.3 fold increase above baseline values) and 1.9 fold for the higher dose of EN. The discrepancy between prothrombin consumption in whole blood and inhibition of thrombin generation rate in PDP in the UH and not in the EN group strongly suggests that UH and not EN is influenced by the presence of a platelet component. This could be formed during thrombin induced platelet activation. Platelet factor 4 is a possible candidate. Another hypothesis involves the role of TFPI-UH complex anticoagulant activity which might be inhibited more during whole blood coagulation than the TFPI-EN complex.
Key words : U.H., LMWH, Anti-X;\,
443
Anti-IIa,
TFPI, Thrombin
generation
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Enoxaparin (EN), a low molecular weight heparin obtained by partial and controlled chemical depolymerization of a benzyl ester of porcine unfractionated heparin, comprises fragments of chains of glycosaminoglycan with a mean molecular weight of about 4,500 Daltons. Clinical development has demonstrated the safety and the efficacy of EN for the prevention of venous thromboembolism in moderate risk-situations such as major general surgery (1) and in high risk situations such as orthopedic surgery of the lower limbs (2,3). Two different dosages (20 and 40 mg) for different levels of risk for venous thromboembolism are used in the prophylaxis of post-operative thrombosis (l-2). A recent study has demonstrated the safety and efficacy of EN using a bid dose regimen of 1 mg/kg body weight (b.w.) via the subcutaneous (KC.) route in the treatment of established DVT (4). The role of Anti-Xa and Anti-IIa activities in the antithrombotic activity of EN has not been clearly established. In addition the inhibition of prothrombinase generation by EN has been demonstrated in vitro (5) as well as ex vivo in patients receiving S.C. injections of 20 mg and 40 mg of EN (6). Indeed, the prothrombin consumption during coagulation of whole blood was significantly altered (6). Tissue factor pathway inhibitor (TFPI) may be implicated in the antithrombotic effect of various glycosaminoglycans (7). It increases after administration of UH and LMWH (8) and inhibits coagulation induced by [Tissue Factor-VIIa] complex (9,lO). It has been claimed that TFPl is a cofactor of heparin and other sulfated polysaccharides (11). TFPI on the other hand is probably the protein which was associated with non purified hepatic triglyceride lipase prepamtions and provides an Anti-Xa clotting activity to these preparations (12). This protein may bind to cell surface (13) and is suspected to be linked to glycosaminoglycans located on the surface of the vessel wall endothelium (14). Its activity decreases with increasing concentrations of phospholipids (15). The objective of this study was to compare in 12 healthy human volunteers the effects of a single S.C. injection of 5,000 IU of UH to a single S.C. injection of 40 mg or 1 mg kg-l b.w. of EN on thrombin generation (using a new automated simple test), as well as Anti-Xa and AntiIIa activities in platelet depleted plasma (PDP). We also measured the effect on prothrombin consumption during coagulation of native whole blood, a more physiological milieu than PDP since platelets can only play a role in the former condition. In addition, tissue factor pathway inhibitor (TFPI) release has been measured. An attempt was made to correlate this activity with Anti-Xa, Anti-IIa, prothrombin consumption in whole blood and thrombin generation in platelet depleted plasma after calcium, tissue thromboplastin and cephalin addition as associated triggers of the coagulation. In this study we demonstrate that a similar inhibition of thrombin generation in platelet depleted plasma is obtained with both heparins. In contrast both EN 40 mg and 1 mg/kg exert a significant inhibitory effect on prothrombin activation during whole blood coagulation which is not found when UH (5,000 IU) is injected. MATERIALS ??
Subjects studied
In a randomized cross over study, twelve healthy male volunteers (23.5 _+4.8 years, 73.0 + 6.4 kg, 180.8 f 5.7 cm) who gave informed written consent received a S.C. injection at one week intervals of 40 mg and 1 mg kg- 1 (73.0 + 6.4 mg) EN and a single dose of 5,000 IU UH. ??
Treatments
Syringes of 5,000 IU of Heparin from Fournier Dijon, France. Prefilled syringes of 40 mg (for 40 mg dose) and vials of 40 mg per 0.4 ml (for 1 mg kg-l treated volunteers) of LMWH (Enoxaparin) were obtained from RhGne-Poulenc Rorer, Antony, France.
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??
Blood sample collection
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TFPI
445
and handling
Blood samples were collected before (0 time) and 0.75, 1.5, 3, 5, 8, 12, 18, 24 hours after sc injection : a) Into Diatubes (from Diagnostica Stago) containing sodium citrate and inhibitors of platelet activation Theophylline, Adenosine, Dipyridamole for all the samples. PDP was obtained as follows : after a first centrifugation at 1,000 rpm at IS’C during 15 mn, the platelet rich plasma is separated. The remaining blood is centrifuged once at 15°C at 4,500 g to obtain platelet poor plasma which is then centrifuged at 4’C at 4,500 g to obtain platelet depleted plasma. PDP was distributed in aliquots of 0.5 ml into previously labeled Eppendorf tubes and immediately frozen at -7O’C for Anti-Xa, Anti-IIa, thrombin generation (TG) and TFPI measurements. These measurements were performed on plasma samples thawed at 37°C. b) A 2 ml blood sample was collected into a glass tube without anticoagulant. Serum was obtained by two centrifugations at 15°C and 3,500 g after two hours incubation time at 37°C and in Eppendorf tubes frozen at -70°C. The residual factor II was measured in this sample.
??
Reagents
- Stachrom Heparin : Bovine factor Xa, CBS 3139 , CH3-S02-D-LEU-GLY-ARG-pCOOH, factor Xa specific substrate. Kit from Diagnostica Stago Asnieres
- Human
NA-
factor IIa from Ortho Diagnostica.
- S 2238 (Factor IIa Specific substrate)
from Kabi, Sweden.
- Factor II deficient plasma and other reagents : human purified factors X and VII, reptilase, rabbit thromboplastin and cephalin (platelet substitute according to Bell and Alton (16) were obtained from Diagnostica Stago, Asnitres. METHODS
Anti-Xa and Anti-IIa activities were measured according to previously published techniques (17). Residual prothrombin in serum was measured using a factor deficient plasma according to a one stage technique (18). Tissue factor pathway inhibitor TFPI was measured according to Sandset technique (19) modified by using CBS 3139 as Xa specific substrate. Briefly, in a first step the following mixture is incubated at 37°C for 25 minutes : 50 1.11Factor VII (0.034 U ml-l), 50 ~1 thromboplastin (0.5 mg ml- I), 50 pl FX (0.025 U ml-l), 50 pl CaC12 (0.075 M), 50 ~1 Plasma l/50 in buffer pH 8 : (Tris-HCl 0.05 M containin g albumin 2 %, Polybrene 2 ktg ml-l, NaCl O.lM, trisodium citrate 0.01 M, NaN3 0.2 %). In a second step, 50 ~1 of FX (0.4 U ml-l) are added to the first mixture in order to measure the residual (Factor VIIa-Tissue Factor) complex activity. After an incubation period of 10 min, the activity of the generated Xa is measured in a third step using 50 ~1 CBS 3 139 3.25 mM and the reaction is stopped after an incubation period of 5 minutes by adding 100 ul of pure acetic acid. The results are expressed as percent of baseline values for each subject. The intra-assay coefficient of variation (c.v.) was 4 % ; the inter assay C.V. was 8 %. Thrombin generation was performed according to a new simplified technique derived from Hemker assay (20) using the automated ACL 300 R apparatus from I.L. Laboratory. Briefly, plasma was defibrinated with reptilase l/20 V/V. To 30 ~1 of defibrinated plasma, 30 l,tl of substrate S 2238 (2 mg ml-l) were added. An incubation during 30 seconds was followed by the addition of 90 ~1 of the reaction mixture consisting of Owren buffer pH 7.35 1 V, CaC12 0.025 M 1 V, cephalin diluted in Owren (l/20) 0,5 V and thromboplastin diluted in Owren l/200 0,5 V. The generated thrombin induced the release of paranitroaniline (PNA) from the ??
??
??
??
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Vol. 69, No. 5
substrate (S 2238). The ACL computer gives the absorbance of PNA at 405 nm at each moment and the time course of the Delta aborbance (A A) was measured every fifteen seconds. This A A increases only when the thrombin amount increases also. A A reaches a “plateau” which corresponds to the maximal amount of generated thrombin. The conversion of A A into thrombin units was obtained using a standard curve performed using human thrombin added at increasing concentrations to defibrinated human plasma. In addition calcium is replaced by buffer in order to avoid endogenous thrombin generation. The results were expressed in Anti-IIa units given by the manufacturer. The rate of thrombin generation was expressed in thrombin U. log min-1. Lag phase and maximal generated thrombin were also measured. The use of human thrombin diluted in the subject defibrinated plasma (obtained before the S.C. drug injection) allowed to measure mainly the additional effect of the drug on the antithrombin activity of the natural inhibitors of thrombin (namely antithrombin III, heparin cof II and ~12 macroglobulin). All the samples of each volunteer were inserted on the same rotor and measured in the same run. The intra assay coefficient of variation (C.V.) for the rate of thrombin generation was ; the C.V. 4.2 % and 3.3 % for control (C) and heparin spiked plasma samples (H) respectively was 11 % and 8 % for the lag phase, for C and H, respectively. The C.V. was 16 % for maximal generated thrombin for C and H. Area under the curve (AUC) values of pharmacodynamic effects as a function of time were calculated using the trapezoidal method. The peak activity (Amax) and its time of appearance (tmax) were taken from the experimental curves. Statistical analysis was performed using the general linear model and the Ryan Einot Gabriel Welsh (REGW) multicomparison test (SAS procedure) (21).
RESULTS
The results are summarized in Tables 1, 11 and 111 and figures 1 to 6 display the time course of Anti-Xa and Anti-Ila activities, prothrombin consumption, inhibition of thrombin generation, lag phase before thrombin generation and TFPI activity in the 3 treatment groups. Table I and fig. 1 show a higher bioavailability of Anti-Xa activity for EN than for UH as evidenced by the AUC values. The mean ratio of Anti-Xa AUC EN/UH was 7 and 15 for EN 40 mg and 1 mg kg-* respectively. Bioavailability (AUCs values) of EN Anti-IIa activity seems higher than that observed after UH since the Anti-IIa EN/UH injected dose ratio (expressed in Anti-IIa units) are 0.25 and 0.45 for the two EN doses respectively (table I and fig. 2). The mean Anti-IIa activity, Amax values were similar for UH 5,000 IU and EN 40 mg (0.06 + 0.02 IU) although the Anti IIa injected doses were 5,000 and 1,200 IU respectively. Moreover, for the two EN treatments, there is a dose dependent increase of Anti-Xa and Anti-IIa activities : a 1.8 fold increase of the injected dose is associated with 1.8 and 2.3 fold increase of maximal Anti-Xa and Anti-IIa activity respectively. Before the injection of each treatment, the baseline values of residual factor II in serum were very low and not significantly different (1.8 + 0.8 % for UH, 1.8 f 1 % for EN 40 and 2.5 f 1.7 % for EN 1 mg kg-l). After injection of UH, the mean peak values of residual factor II were significantly lower (7 and 12 fold) than those observed after EN 40 mg and EN 1 mg kg-l, respectively. The area under the curve values, also show a striking difference between UH and EN groups (Table I). UH injection in contrast to EN is not associated with a significant increase of residual factor II in serum as demonstrated by the observed mean Amax and AUC values. Figure 4 emphasizes the dose dependent inhibition of prothrombin consumption for EN, with a maximal activity around three hours.
447
HEPARINS, FII CONSUMPTION, TFPI
vol. 69, No. 5
r\NTI
X,
FACTOR
ACTIVITY
II CCNSIJMPTION
TFPI
ANTI I$ ACTIVITY
LAGPHASE
FIG.
1-6.
Time course of Anti-Xa (Fig. l), Anti-IIa activity (Fig. 2), thrombin generation rate inhibition (Fig. 3) prothrombin consumption (Fig. 4) release of tissue factorpathway inhibitor (Fig. 5) and lag phase of thrombin generation (Fig. 6) after subcutaneous injection of EN+ 40 mgoe, or 1 mg kg- I-, or UH 5,000 IUCrat one week interval.
448
HEPARINS, FII CONSUMPTION,
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Vol. 69, No. 5
TABLE I COMPARISON OF AUC AND PEAK VALUES (A max) FOR ANTI-Xa, ANTI-IIa ACTIVITIES AND RESIDUAL FACTOR II IN SERUM AFTER SUB-CUTANEOUS INJECTION OF UH 5,000 IU, EN 40 mg and1 mg kg- 1 I
UH
EN 40 mg
EN 1 mgKg-1
Injected Doses Anti-Xa (IU) Anti-IIa (IU)
5,000 5,000
4,000 1,200
7,300 f 640 2,190 + 192
4 maxi (peak activity) Anti-Xa (IU) Anti-IIa (IU) Residual F II (%)
0.06 f 0.02 0.06 _+0.03 2.46 f 1.20
0.40 + 0.04 0.06 _+0.02 17.50 + 4.90
0.73 f 0.14 0.14+ 0.05 30.00 f 11.oo
WC2 Anti-Xa (h.IU.mL-1) Anti-IIa (h.IU.mL-1) Residual FII (%.h-1)
0.47 f 0.30 0.34 t- 0.24 6.10 +_20.0
3.35 * 0.38 0.44+ 0.18 127.00 +_66.40
7.10 f 1.10 1.06 f 0.37 277.70 + 124.40 d
2 = AUC : Area under the curve [activity = f (time)] 1 = A Max : Maximal activity Data are given as mean + one standard deviation (n = 12). All the differences observed in the results of the 3 treatment groups are statistically different (p < 0.05) except for Anti-IIa, A max and AUC which are similar for UH 5,000 IU and EN 40 mg. TABLE II COMPARISON OF MAXIMAL ACTIVITIES OF THE INHIBITION OF THROMBIN GENERATION RATE AND TFPI RELEASE AFTER S.C INJECTION OF UH 5,000 IL’, EN 40 MC AND EN 1 mg/kg-l
n
Heparin
b) Inhibition %
8
23.6 + 14.7
17.0 + 7.2
30.0 + 6.7
D)Lag phase (LP) Ratio LP Max /LP baseline
8
1.5 & 0.4
1.31 0.2
1.5+, 0.2
TFPI
8
140.0 f 29.0
140.0 + 25
193.0 + 46
EN 40 mg
EN 1 mgKg-1
rhrombin generation
% vs baseline values
Data are given as mean + SD (n = 8 subjects)
HEPARINS. FII CONSUMPTION,
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TFPI
449
A significant inhibition of thrombin generation rate (TG) is evidenced with the 3 treatment groups as compared to basal values (p < 0,05) (Table II). It is similar for UH and EN 40 mg (23.6 k 14.7 %, and 17 + 7.2 % respectively) and significantly higher for the EN 1 mg kg-l injected dose (30.0 f 6.7 %) p < 0.001. Figures 3 and 6 show a maximal thrombin generation rate inhibition 90 minutes after EN 1 mg kg-l whereas after EN (40 mg) and UH, this rate inhibitory effect occurs at the third hour. Table II shows a similar peak for TFPI release [ 1.40 fold as compared to baseline values] for UH and EN 40 mg (p = 0,005). After the injection of 1 mg kg-l of EN, TFPI maximal activity is 1.93 fold that of the plasma control before the injection of the drug and significantly higher (p = 0.02) than after subcutaneous injection of EN 40. The maximal activities are reached about 90 minutes after S.C. injection ( fig. 5). TABLE INHIBITION
OF TOTAL
Ill
GENERATED
THROMBIN
% inhibition at 45 min. UH
19.1
EN 40 mg
16.56 f. 10.7
EN 1 mg kg-l
34.28 f 2 1
*
s : (p
% inhibition at 90 min.
f 17.8
10.6 NS
(%)
*
+
5.8
10.45 * 11 23.7
NS
**
f 11
= 0.05 ) ** s : (p = 0.02)
Total generated thrombin (Table Ill) was not significantly modified in UH 5,000 IU and EN 40 mg treated volunteers (baseline value = 0%) while a significant inhibitory effect of 34 % (p = 0.02) and 23.7%+11% (p = 0.05) was observed 45 and 90 minutes after S.C. injection of EN 1 mg kg-l. In summary, whatever the treatment, maximal activity for TFPI release was observed 90 minutes after SC injection. It was about 180 minutes for thrombin generation inhibition, prothrombin consumption, Anti-Xa, Anti-lla activities. However at the higher EN dose, thrombin generation rate inhibition and reduction of generated thrombin occured earlier : Tmax = 90 minutes. DISCUSSION - The results of this work confirm the higher bioavailability of Anti-Xa activity after EN S.C. injection in volunteers as compared to UH SC injection of 5,000 IU (17). Interestingly, similar Anti-lla activity was obtained in UH and EN 40 mg groups (peak activity and area under the curve) although the Anti-lla injected doses are four fold greater for UH (5,000 IU) than for EN (1,200 IU) (Table I). - The increase of TFPI activity release was similar (1.40 fold versus baseline values for both UH and EN 40 mg) while Anti-Xa activity was much higher in the EN 40 group than in the UH group. These results could suggest that TFPI release is generated by fragments other than those responsible for sole Anti-Xu activity. The identification of the nature of these fragments involved in the TFPI release requires further investigation. The increase of TFPI activity after UH and EN 40 mg was similar (23.6 + 14.7 % and 17.0 -t 7.2 % respectively). It was associated with a comparable inhibition of the thrombin generation in platelet depleted plasma. In contrast
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TFPI
Vol. 69, No. 5
the similar increase of TFPI activity following EN 40 and UH 5,000 treatments was associated with a clearly different inhibition of prothrombin consumption. Moreover, UH and LMWH, when added to PDP in vitro, exert a lower inhibitory activity on thrombin generation after thromboplastin + cephalin + Ca triggered coagulation (automated technique) than in ex vivo samples (results not shown). These results are in accordance with recently published papers suggesting that the inhibition of thrombin generation in vivo is influenced by the release of TFPI (9,lO). - It is important to note that in whole blood, prothrombin consumption is inhibited in both EN treatments but not in the UH treatment group. Perhaps increasing the dose of UH could have resulted in higher levels of residual prothrombin. Nevertheless the important difference regarding prothrombin consumption observed in EN 40 and UH 5,000 groups contrasts with the similar inhibition of thrombin generation observed in PDP in these treatment groups. Apparently this discrepancy is linked to the fact that prothrombin activation occurs in different conditions in these tests : in the prothrombin consumption assay prothrombin is activated to thrombin in whole blood where platelets and calcium are present in their normal milieu. In contrast the thrombin generation test is performed in titrated platelet depleted plasma. Thus this discrepancy could involve platelets. Several explanations can be proposed : a) An inhibitory effect of platelets present in whole blood and not in PDP : on the one hand platelet factor 4 is released during thrombin induced platelet activation and has a more potent inhibitory effect on UH than on EN (22-24). On the other hand, it has been suggested that the protection of factor Xa associated with platelet membrane phospholipids is greater with UH than with LMWH (2526). b) The higher Anti-Xa activity exhibited in the EN groups as compared to UH group could be responsible for the greater inhibition of prothrombin activation. However the similar inhibitory activity on thrombin generation in PDP observed in UH group and EN 40 mg group more likely suggests that Anti-Xa concentration does not play a major role in this test. Thus, the different effect of activated platelets on UH and on EN seems to be a better candidate than AntiXa activity for the explanation of this discrepancy. c) A more likely hypothesis could be the existence of a greater inhibitory effect of activated platelets on TFPI-UH complexes than on TFPI-EN complexes. Finally, taking into account the similar antithrombotic activity of EN 40 mg and UH 5,000 Anti-Xa IU, as demonstrated in clinical trials (1) it is difficult to understand why EN 40 in contrast to UH 5,000 IU is associated with an inhibition of prothrombin consumption in whole blood during coagulation. Thus, at these effective prophylactic doses, UH and EN 40 mg could exert their antithrombotic activity at least partially through a different mechanism of action which involves for instance the vessel endothelial wall binding of similar UH and EN fragments. The results suggest that inhibition of prothrombin consumption may not be clinically relevant for prophylactic treatment with UH. Its relevance in patients receiving LMWH warrants further investigation.
1. SAMAMA, M., BERNARD, P. ,BONNARDOT, J.P., COMBE-TAMZALI, S., LANSON, Y. and TISSOT, E. Low molecular weight hepsrin compared with unfractionated heparin in prevention of post-operative thrombosis. Br. J. Surg. 75,128-131, 1988. 2. PLANES, A., VOCHELLE, N. MAZAS F., MANSAT, C., ZUCMAN, J., LANDAIS, A., PASCARIELLO, J.C., WEILL, D., and BUTEL, J.Prevention of post-operative venous thrombosis : a randomized trial comparing unfractionated heparin with low molecular weight heparin in patients undergoin, ~7total hip replacement. Thromhos. Huemost. 60 (3), 407-410, 1988. 3. TURPIE, A.G.G., LEVINE, M. and HIRSH J. A randomized controlled trial of a low molecular weight heparin (Enoxaparin) to prevent deep vein thrombosis in patients undergoing elective hip surgery. N. Engl. J. Med. 315,925-929, 1986.
Vol. 69, No. 5
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4. SIMONNEAU, G. for the TVPENOX group Study. Subcutaneous fixed dose of enoxaparin (E) versus intravenous adjusted dose of unfractionated heparin (UH) in the treatment of deep venous thrombosis (DVT). Thrombos. Haemost. 65 (6), 754,306a, 1991. 5. HEMKER H.C. The mode of action of heparin in plasma. In : Thrombosis and Haemostasis. Verstraete M., Vermylen J. Lijnen R, Amout N. (Ed). Leuven University 1987, pp. 17-32. 6. SAMAMA, M.M., COMBE, S., HORELLOU, M.H., AUGRAS, D., TRUC, J.B., CONARD, J., GIRARD, P., BARA, L. and POITOUT, PH. Anti-Xa activity and prothrombinase inhibition in patients treated with two different doses of enoxaparin in gynecologic surgery. Thrombos. Res. suppl. XIV, 29-37, 1991. 7. SANDSET, L. and ABILDGAARD, U. Extrinsic pathway inhibitor. The key of blood coagulation initiated by tissue thromboplastin. Haemosrasis 21, 219-239, 1991. 8. SANDSET, L., ABILDGAARD, coagulation pathway inhibitor
U. and LARSEN B.L. Heparin induces release of extrinsic (EPI). Thrombos. Rrs. 50, 803-813, 1988.
9. LINDAHL, A.K., ABILDGAARD, U., LARSEN, B.L., STAALESEN, R., GANGNAESHAMMER, A.K., SANDSET, P.M., NORDFANG, 0. and BECK, T.C. Extrinsic pathway inhibitor (EPI) released to the blood by heparin is a more powerful coagulation inhibitor than recombinant EPI. Thromhos. Res. 62, 607-614, 1991. 10. LINDAHL, A.K., ABILDGAARD, U., LARSEN, B.L., AAMODT, LM., NORDFANG, 0. and BECK, T.C. Extrinsic pathway inhibitor (EPI) and post heparin anticoagulant effect in tissue thromboplasin induced coagulation. Thromhos. Res. suppl. XIV,39-48, 1991. 11. WUN-TZE, Chein. Lipoprotein associated coagulation inhibitor (LACI) is a cofactor for heparin ; synergistic anticoagulant action between LACI and sulfated polysaccharides. Blood 79,430-438, 1992. 12. SCHMIDT, M., BARROWCLIFFE, T.W., GRAY, E., WAITON, G. and HARENBERG, Anti-Xa clotting activities in different hepatic triglyceride lipase preparations from post heparin plasma. Thromhos. Res. 63, 503-508, 199 1. 13. CALLENDER, N.S., RAO, L.V.M., SANDSER, P.M., WARNCRAMER, B.J. and RAPAPORT, S.1. Binding studies of extrinsic pathway inhibitor (EPI) to a cell surface. Blood 76 (suppl. I), 414 a, 1990. 14. NOVOTNY, F. W., PALMIER, M., WUN, T.C., BROZE, J.G. Jr and MILETICH, Purification and properties of heparin releasable lipoprotein associated coagulation inhibitor. Blood 78, 2, 394-400, 1991.
J.P.
15. BROZE, G.J.Jr., WARREN L.A., NOVOTNY, W.F., HIGUCHI, D.A., GIRAR, J.J., MILETICH, J.P. The lipoprotein-associated coagulation inhibitor that inhibit the factor VII-Tissue factor complex, also inhibits factor Xa : insight into its possible mechanism of action. Bloc,d 71, (3), 335-333, 1988. 16. BELL, W.N., ALTON H.G. A brain extract as a substitute for platelet suspensions thromboplastin generation test. Narwe 174, 880-88 1, 1954.
in the
17. BARA, L., BILLAUD, E., GRAMOND, G., KHER, A. and SAMAMA M. Comparative pharmacokinetics of a low molecular weight heparin (PK 10169) and unfractionated heparin after intravenous and subcutaneous administration. Thrombos. Res. 39, 631-636, 1985.
J.
452
HEPARINS, FII CONSUMPTION, TFPI
Vol. 69, No. 5
18. CAEN, J., LARRIEU, M.Y. and SAMAMA, M. La consommation de prothrombine. In :
I’Htmostase. Methodes d’exploration et diagnostic pratique (Ed.) L’expansion Scientifique (Paris) deuxieme edition, 1975, pp. 173-176. 19. SANDSET, P.M., ABILDGAARD, U. and PETTERSEN, M. A sensitive assay of extrinsic coagulation pathway inhibitor (EPI in plasma and plasma fractions). Thrombos. Res. 47, 389-400,1987.
20. HEMKER, H.C., WILLEMS, G.M., BEGUIN, S. A computer assisted method to obtain the
prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb. Haemost. 56,9-17, 1986. 21. SAS (Statistical Analysis System) procedure. SAS Institute INC. Box 8000 Cary, North Carolina 275 11, U.S.A. 22. LANE, D.A., DENTON, J., FLYNN, A.M., THUNBERG, L. and LINDAHL, U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem. J. 218,725-732, 1984. 23. LANE, D.A., PEJLER, G., FLYNN, A.M., THOMPSON, E.A. and LINDAHL, U. Neutralization of heparin - related saccharides by histidine-rich glycoprotein and platelet factor 4. J. Biol. Chem. 261,3980-3986, 1986. 24. RUCINSKI, B., NIEWIAROWSKI, S, STRZYZEWSKI, M. HOLT, J.C. and MAYO, K.H. Platelet factor 4 and its C terminal human peptides : heparin binding and clearance from the circulation. Thrombos Haemost. 63, (3), 493-498, 1990. 25. BEGHIN, S., MARDIGUIAN, J., LINDHOUT, T. and HEMKER, H.C. The mode of action
of low molecular weight heparin preparation (PK 10169) and two of its major components on thrombin generation in plasma. Thrombos Haemost. 61 (l), 30-34, 1989. 26. ELLIS, V., SCULLY, M.F. and KAKKAR V.V. The acceleration of the inhibition of prothrombinase complex by heparin. Biochem. J. 233, 161-165, 1986.
COMPARATIVE EFFECTS OF ENOXAPARIN AND UNFRACTIONATED HEPARIN IN HEALTHY VOLUNTEERS ON PROTHROMBIN CONSUMPTION IN WHOLE BLOOD
DURING
COAGULATION, PATHWAY
AND RELEASE INHIBITOR
OF TISSUE
FACTOR
L. Bara*, M.F. Bloch*, D. Zitoun”, M. &mama**, F. Collignon***, A. Frydman ***, A. Uzan***, J. Bouthier***. “Laboratoire de Thrombose Expkimentale, Universitk Pierre et Marie Curie, Paris, France,** Laboratoire Central d’Hkmatologie, Hbtel-Dieu, Paris, France, *** RhGnePoulenc Rorer, Recherche et DCveloppement, Antony, France. (Received 10.7.1992; accepted in revised form 23.12.1992 by Editor D.A. Lane)
ABSTRACT In a randomized crossover study twelve healthy male volunteers (23.5 + of 4.8 years, 73.0 + 6.4 kg, 180.8 f 5.7 cm) received one subcutaneous injection of either enoxaparin (EN) at 40 mg or 1 mg kg-l, or unfractionated heparin (UH) at 5,000 IU at one week intervals. Area under curves (AUC) of Anti-Xa and Anti-IIa activities correlated with EN dose. The relative effectiveness of EN versus UH 5,000 U as assessed by AUC ratio (EN/UH) was 7 and 15 for Anti-Xa activity, 1.3 and 3.1 for Anti-IIa activity after SC injection of EN 40 mg (4,000 Anti-Xa IU and 1,200 Anti-IIa’U) and 1 mg kg-l (7,300 + 640 Anti-Xa 1U and 2,190 + 290 Anti-IIa IU) respectively. In volunteers receiving EN, a dose dependent inhibition of thrombin generation rate in platelet depleted plasma (PDP), measured with a new and simple chromogenic thrombin generation assay, was observed when compared with baseline values. Similarly, intrinsic prothrombin activation in whole blood, evidenced by measuring residual factor II in serum 2 hours after clotting (prothrombin consumption test : PC), was inhibited in a dose dependent manner. In UH treated volunteers, although the inhibition of thrombin generation rate in PDP was similar to that observed with EN 40 mg, prothrombin consumption in whole blood was not significantly modified. Tissue factor pathway inhibitor (TFPI) activity release was increased similarly for UH and EN 40 ( 1.3 fold increase above baseline values) and 1.9 fold for the higher dose of EN. The discrepancy between prothrombin consumption in whole blood and inhibition of thrombin generation rate in PDP in the UH and not in the EN group strongly suggests that UH and not EN is influenced by the presence of a platelet component. This could be formed during thrombin induced platelet activation. Platelet factor 4 is a possible candidate. Another hypothesis involves the role of TFPI-UH complex anticoagulant activity which might be inhibited more during whole blood coagulation than the TFPI-EN complex.
Key words : U.H., LMWH, Anti-X;\,
443
Anti-IIa,
TFPI, Thrombin
generation
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Enoxaparin (EN), a low molecular weight heparin obtained by partial and controlled chemical depolymerization of a benzyl ester of porcine unfractionated heparin, comprises fragments of chains of glycosaminoglycan with a mean molecular weight of about 4,500 Daltons. Clinical development has demonstrated the safety and the efficacy of EN for the prevention of venous thromboembolism in moderate risk-situations such as major general surgery (1) and in high risk situations such as orthopedic surgery of the lower limbs (2,3). Two different dosages (20 and 40 mg) for different levels of risk for venous thromboembolism are used in the prophylaxis of post-operative thrombosis (l-2). A recent study has demonstrated the safety and efficacy of EN using a bid dose regimen of 1 mg/kg body weight (b.w.) via the subcutaneous (KC.) route in the treatment of established DVT (4). The role of Anti-Xa and Anti-IIa activities in the antithrombotic activity of EN has not been clearly established. In addition the inhibition of prothrombinase generation by EN has been demonstrated in vitro (5) as well as ex vivo in patients receiving S.C. injections of 20 mg and 40 mg of EN (6). Indeed, the prothrombin consumption during coagulation of whole blood was significantly altered (6). Tissue factor pathway inhibitor (TFPI) may be implicated in the antithrombotic effect of various glycosaminoglycans (7). It increases after administration of UH and LMWH (8) and inhibits coagulation induced by [Tissue Factor-VIIa] complex (9,lO). It has been claimed that TFPl is a cofactor of heparin and other sulfated polysaccharides (11). TFPI on the other hand is probably the protein which was associated with non purified hepatic triglyceride lipase prepamtions and provides an Anti-Xa clotting activity to these preparations (12). This protein may bind to cell surface (13) and is suspected to be linked to glycosaminoglycans located on the surface of the vessel wall endothelium (14). Its activity decreases with increasing concentrations of phospholipids (15). The objective of this study was to compare in 12 healthy human volunteers the effects of a single S.C. injection of 5,000 IU of UH to a single S.C. injection of 40 mg or 1 mg kg-l b.w. of EN on thrombin generation (using a new automated simple test), as well as Anti-Xa and AntiIIa activities in platelet depleted plasma (PDP). We also measured the effect on prothrombin consumption during coagulation of native whole blood, a more physiological milieu than PDP since platelets can only play a role in the former condition. In addition, tissue factor pathway inhibitor (TFPI) release has been measured. An attempt was made to correlate this activity with Anti-Xa, Anti-IIa, prothrombin consumption in whole blood and thrombin generation in platelet depleted plasma after calcium, tissue thromboplastin and cephalin addition as associated triggers of the coagulation. In this study we demonstrate that a similar inhibition of thrombin generation in platelet depleted plasma is obtained with both heparins. In contrast both EN 40 mg and 1 mg/kg exert a significant inhibitory effect on prothrombin activation during whole blood coagulation which is not found when UH (5,000 IU) is injected. MATERIALS ??
Subjects studied
In a randomized cross over study, twelve healthy male volunteers (23.5 _+4.8 years, 73.0 + 6.4 kg, 180.8 f 5.7 cm) who gave informed written consent received a S.C. injection at one week intervals of 40 mg and 1 mg kg- 1 (73.0 + 6.4 mg) EN and a single dose of 5,000 IU UH. ??
Treatments
Syringes of 5,000 IU of Heparin from Fournier Dijon, France. Prefilled syringes of 40 mg (for 40 mg dose) and vials of 40 mg per 0.4 ml (for 1 mg kg-l treated volunteers) of LMWH (Enoxaparin) were obtained from RhGne-Poulenc Rorer, Antony, France.
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??
Blood sample collection
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445
and handling
Blood samples were collected before (0 time) and 0.75, 1.5, 3, 5, 8, 12, 18, 24 hours after sc injection : a) Into Diatubes (from Diagnostica Stago) containing sodium citrate and inhibitors of platelet activation Theophylline, Adenosine, Dipyridamole for all the samples. PDP was obtained as follows : after a first centrifugation at 1,000 rpm at IS’C during 15 mn, the platelet rich plasma is separated. The remaining blood is centrifuged once at 15°C at 4,500 g to obtain platelet poor plasma which is then centrifuged at 4’C at 4,500 g to obtain platelet depleted plasma. PDP was distributed in aliquots of 0.5 ml into previously labeled Eppendorf tubes and immediately frozen at -7O’C for Anti-Xa, Anti-IIa, thrombin generation (TG) and TFPI measurements. These measurements were performed on plasma samples thawed at 37°C. b) A 2 ml blood sample was collected into a glass tube without anticoagulant. Serum was obtained by two centrifugations at 15°C and 3,500 g after two hours incubation time at 37°C and in Eppendorf tubes frozen at -70°C. The residual factor II was measured in this sample.
??
Reagents
- Stachrom Heparin : Bovine factor Xa, CBS 3139 , CH3-S02-D-LEU-GLY-ARG-pCOOH, factor Xa specific substrate. Kit from Diagnostica Stago Asnieres
- Human
NA-
factor IIa from Ortho Diagnostica.
- S 2238 (Factor IIa Specific substrate)
from Kabi, Sweden.
- Factor II deficient plasma and other reagents : human purified factors X and VII, reptilase, rabbit thromboplastin and cephalin (platelet substitute according to Bell and Alton (16) were obtained from Diagnostica Stago, Asnitres. METHODS
Anti-Xa and Anti-IIa activities were measured according to previously published techniques (17). Residual prothrombin in serum was measured using a factor deficient plasma according to a one stage technique (18). Tissue factor pathway inhibitor TFPI was measured according to Sandset technique (19) modified by using CBS 3139 as Xa specific substrate. Briefly, in a first step the following mixture is incubated at 37°C for 25 minutes : 50 1.11Factor VII (0.034 U ml-l), 50 ~1 thromboplastin (0.5 mg ml- I), 50 pl FX (0.025 U ml-l), 50 pl CaC12 (0.075 M), 50 ~1 Plasma l/50 in buffer pH 8 : (Tris-HCl 0.05 M containin g albumin 2 %, Polybrene 2 ktg ml-l, NaCl O.lM, trisodium citrate 0.01 M, NaN3 0.2 %). In a second step, 50 ~1 of FX (0.4 U ml-l) are added to the first mixture in order to measure the residual (Factor VIIa-Tissue Factor) complex activity. After an incubation period of 10 min, the activity of the generated Xa is measured in a third step using 50 ~1 CBS 3 139 3.25 mM and the reaction is stopped after an incubation period of 5 minutes by adding 100 ul of pure acetic acid. The results are expressed as percent of baseline values for each subject. The intra-assay coefficient of variation (c.v.) was 4 % ; the inter assay C.V. was 8 %. Thrombin generation was performed according to a new simplified technique derived from Hemker assay (20) using the automated ACL 300 R apparatus from I.L. Laboratory. Briefly, plasma was defibrinated with reptilase l/20 V/V. To 30 ~1 of defibrinated plasma, 30 l,tl of substrate S 2238 (2 mg ml-l) were added. An incubation during 30 seconds was followed by the addition of 90 ~1 of the reaction mixture consisting of Owren buffer pH 7.35 1 V, CaC12 0.025 M 1 V, cephalin diluted in Owren (l/20) 0,5 V and thromboplastin diluted in Owren l/200 0,5 V. The generated thrombin induced the release of paranitroaniline (PNA) from the ??
??
??
??
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substrate (S 2238). The ACL computer gives the absorbance of PNA at 405 nm at each moment and the time course of the Delta aborbance (A A) was measured every fifteen seconds. This A A increases only when the thrombin amount increases also. A A reaches a “plateau” which corresponds to the maximal amount of generated thrombin. The conversion of A A into thrombin units was obtained using a standard curve performed using human thrombin added at increasing concentrations to defibrinated human plasma. In addition calcium is replaced by buffer in order to avoid endogenous thrombin generation. The results were expressed in Anti-IIa units given by the manufacturer. The rate of thrombin generation was expressed in thrombin U. log min-1. Lag phase and maximal generated thrombin were also measured. The use of human thrombin diluted in the subject defibrinated plasma (obtained before the S.C. drug injection) allowed to measure mainly the additional effect of the drug on the antithrombin activity of the natural inhibitors of thrombin (namely antithrombin III, heparin cof II and ~12 macroglobulin). All the samples of each volunteer were inserted on the same rotor and measured in the same run. The intra assay coefficient of variation (C.V.) for the rate of thrombin generation was ; the C.V. 4.2 % and 3.3 % for control (C) and heparin spiked plasma samples (H) respectively was 11 % and 8 % for the lag phase, for C and H, respectively. The C.V. was 16 % for maximal generated thrombin for C and H. Area under the curve (AUC) values of pharmacodynamic effects as a function of time were calculated using the trapezoidal method. The peak activity (Amax) and its time of appearance (tmax) were taken from the experimental curves. Statistical analysis was performed using the general linear model and the Ryan Einot Gabriel Welsh (REGW) multicomparison test (SAS procedure) (21).
RESULTS
The results are summarized in Tables 1, 11 and 111 and figures 1 to 6 display the time course of Anti-Xa and Anti-Ila activities, prothrombin consumption, inhibition of thrombin generation, lag phase before thrombin generation and TFPI activity in the 3 treatment groups. Table I and fig. 1 show a higher bioavailability of Anti-Xa activity for EN than for UH as evidenced by the AUC values. The mean ratio of Anti-Xa AUC EN/UH was 7 and 15 for EN 40 mg and 1 mg kg-* respectively. Bioavailability (AUCs values) of EN Anti-IIa activity seems higher than that observed after UH since the Anti-IIa EN/UH injected dose ratio (expressed in Anti-IIa units) are 0.25 and 0.45 for the two EN doses respectively (table I and fig. 2). The mean Anti-IIa activity, Amax values were similar for UH 5,000 IU and EN 40 mg (0.06 + 0.02 IU) although the Anti IIa injected doses were 5,000 and 1,200 IU respectively. Moreover, for the two EN treatments, there is a dose dependent increase of Anti-Xa and Anti-IIa activities : a 1.8 fold increase of the injected dose is associated with 1.8 and 2.3 fold increase of maximal Anti-Xa and Anti-IIa activity respectively. Before the injection of each treatment, the baseline values of residual factor II in serum were very low and not significantly different (1.8 + 0.8 % for UH, 1.8 f 1 % for EN 40 and 2.5 f 1.7 % for EN 1 mg kg-l). After injection of UH, the mean peak values of residual factor II were significantly lower (7 and 12 fold) than those observed after EN 40 mg and EN 1 mg kg-l, respectively. The area under the curve values, also show a striking difference between UH and EN groups (Table I). UH injection in contrast to EN is not associated with a significant increase of residual factor II in serum as demonstrated by the observed mean Amax and AUC values. Figure 4 emphasizes the dose dependent inhibition of prothrombin consumption for EN, with a maximal activity around three hours.
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r\NTI
X,
FACTOR
ACTIVITY
II CCNSIJMPTION
TFPI
ANTI I$ ACTIVITY
LAGPHASE
FIG.
1-6.
Time course of Anti-Xa (Fig. l), Anti-IIa activity (Fig. 2), thrombin generation rate inhibition (Fig. 3) prothrombin consumption (Fig. 4) release of tissue factorpathway inhibitor (Fig. 5) and lag phase of thrombin generation (Fig. 6) after subcutaneous injection of EN+ 40 mgoe, or 1 mg kg- I-, or UH 5,000 IUCrat one week interval.
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TABLE I COMPARISON OF AUC AND PEAK VALUES (A max) FOR ANTI-Xa, ANTI-IIa ACTIVITIES AND RESIDUAL FACTOR II IN SERUM AFTER SUB-CUTANEOUS INJECTION OF UH 5,000 IU, EN 40 mg and1 mg kg- 1 I
UH
EN 40 mg
EN 1 mgKg-1
Injected Doses Anti-Xa (IU) Anti-IIa (IU)
5,000 5,000
4,000 1,200
7,300 f 640 2,190 + 192
4 maxi (peak activity) Anti-Xa (IU) Anti-IIa (IU) Residual F II (%)
0.06 f 0.02 0.06 _+0.03 2.46 f 1.20
0.40 + 0.04 0.06 _+0.02 17.50 + 4.90
0.73 f 0.14 0.14+ 0.05 30.00 f 11.oo
WC2 Anti-Xa (h.IU.mL-1) Anti-IIa (h.IU.mL-1) Residual FII (%.h-1)
0.47 f 0.30 0.34 t- 0.24 6.10 +_20.0
3.35 * 0.38 0.44+ 0.18 127.00 +_66.40
7.10 f 1.10 1.06 f 0.37 277.70 + 124.40 d
2 = AUC : Area under the curve [activity = f (time)] 1 = A Max : Maximal activity Data are given as mean + one standard deviation (n = 12). All the differences observed in the results of the 3 treatment groups are statistically different (p < 0.05) except for Anti-IIa, A max and AUC which are similar for UH 5,000 IU and EN 40 mg. TABLE II COMPARISON OF MAXIMAL ACTIVITIES OF THE INHIBITION OF THROMBIN GENERATION RATE AND TFPI RELEASE AFTER S.C INJECTION OF UH 5,000 IL’, EN 40 MC AND EN 1 mg/kg-l
n
Heparin
b) Inhibition %
8
23.6 + 14.7
17.0 + 7.2
30.0 + 6.7
D)Lag phase (LP) Ratio LP Max /LP baseline
8
1.5 & 0.4
1.31 0.2
1.5+, 0.2
TFPI
8
140.0 f 29.0
140.0 + 25
193.0 + 46
EN 40 mg
EN 1 mgKg-1
rhrombin generation
% vs baseline values
Data are given as mean + SD (n = 8 subjects)
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TFPI
449
A significant inhibition of thrombin generation rate (TG) is evidenced with the 3 treatment groups as compared to basal values (p < 0,05) (Table II). It is similar for UH and EN 40 mg (23.6 k 14.7 %, and 17 + 7.2 % respectively) and significantly higher for the EN 1 mg kg-l injected dose (30.0 f 6.7 %) p < 0.001. Figures 3 and 6 show a maximal thrombin generation rate inhibition 90 minutes after EN 1 mg kg-l whereas after EN (40 mg) and UH, this rate inhibitory effect occurs at the third hour. Table II shows a similar peak for TFPI release [ 1.40 fold as compared to baseline values] for UH and EN 40 mg (p = 0,005). After the injection of 1 mg kg-l of EN, TFPI maximal activity is 1.93 fold that of the plasma control before the injection of the drug and significantly higher (p = 0.02) than after subcutaneous injection of EN 40. The maximal activities are reached about 90 minutes after S.C. injection ( fig. 5). TABLE INHIBITION
OF TOTAL
Ill
GENERATED
THROMBIN
% inhibition at 45 min. UH
19.1
EN 40 mg
16.56 f. 10.7
EN 1 mg kg-l
34.28 f 2 1
*
s : (p
% inhibition at 90 min.
f 17.8
10.6 NS
(%)
*
+
5.8
10.45 * 11 23.7
NS
**
f 11
= 0.05 ) ** s : (p = 0.02)
Total generated thrombin (Table Ill) was not significantly modified in UH 5,000 IU and EN 40 mg treated volunteers (baseline value = 0%) while a significant inhibitory effect of 34 % (p = 0.02) and 23.7%+11% (p = 0.05) was observed 45 and 90 minutes after S.C. injection of EN 1 mg kg-l. In summary, whatever the treatment, maximal activity for TFPI release was observed 90 minutes after SC injection. It was about 180 minutes for thrombin generation inhibition, prothrombin consumption, Anti-Xa, Anti-lla activities. However at the higher EN dose, thrombin generation rate inhibition and reduction of generated thrombin occured earlier : Tmax = 90 minutes. DISCUSSION - The results of this work confirm the higher bioavailability of Anti-Xa activity after EN S.C. injection in volunteers as compared to UH SC injection of 5,000 IU (17). Interestingly, similar Anti-lla activity was obtained in UH and EN 40 mg groups (peak activity and area under the curve) although the Anti-lla injected doses are four fold greater for UH (5,000 IU) than for EN (1,200 IU) (Table I). - The increase of TFPI activity release was similar (1.40 fold versus baseline values for both UH and EN 40 mg) while Anti-Xa activity was much higher in the EN 40 group than in the UH group. These results could suggest that TFPI release is generated by fragments other than those responsible for sole Anti-Xu activity. The identification of the nature of these fragments involved in the TFPI release requires further investigation. The increase of TFPI activity after UH and EN 40 mg was similar (23.6 + 14.7 % and 17.0 -t 7.2 % respectively). It was associated with a comparable inhibition of the thrombin generation in platelet depleted plasma. In contrast
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the similar increase of TFPI activity following EN 40 and UH 5,000 treatments was associated with a clearly different inhibition of prothrombin consumption. Moreover, UH and LMWH, when added to PDP in vitro, exert a lower inhibitory activity on thrombin generation after thromboplastin + cephalin + Ca triggered coagulation (automated technique) than in ex vivo samples (results not shown). These results are in accordance with recently published papers suggesting that the inhibition of thrombin generation in vivo is influenced by the release of TFPI (9,lO). - It is important to note that in whole blood, prothrombin consumption is inhibited in both EN treatments but not in the UH treatment group. Perhaps increasing the dose of UH could have resulted in higher levels of residual prothrombin. Nevertheless the important difference regarding prothrombin consumption observed in EN 40 and UH 5,000 groups contrasts with the similar inhibition of thrombin generation observed in PDP in these treatment groups. Apparently this discrepancy is linked to the fact that prothrombin activation occurs in different conditions in these tests : in the prothrombin consumption assay prothrombin is activated to thrombin in whole blood where platelets and calcium are present in their normal milieu. In contrast the thrombin generation test is performed in titrated platelet depleted plasma. Thus this discrepancy could involve platelets. Several explanations can be proposed : a) An inhibitory effect of platelets present in whole blood and not in PDP : on the one hand platelet factor 4 is released during thrombin induced platelet activation and has a more potent inhibitory effect on UH than on EN (22-24). On the other hand, it has been suggested that the protection of factor Xa associated with platelet membrane phospholipids is greater with UH than with LMWH (2526). b) The higher Anti-Xa activity exhibited in the EN groups as compared to UH group could be responsible for the greater inhibition of prothrombin activation. However the similar inhibitory activity on thrombin generation in PDP observed in UH group and EN 40 mg group more likely suggests that Anti-Xa concentration does not play a major role in this test. Thus, the different effect of activated platelets on UH and on EN seems to be a better candidate than AntiXa activity for the explanation of this discrepancy. c) A more likely hypothesis could be the existence of a greater inhibitory effect of activated platelets on TFPI-UH complexes than on TFPI-EN complexes. Finally, taking into account the similar antithrombotic activity of EN 40 mg and UH 5,000 Anti-Xa IU, as demonstrated in clinical trials (1) it is difficult to understand why EN 40 in contrast to UH 5,000 IU is associated with an inhibition of prothrombin consumption in whole blood during coagulation. Thus, at these effective prophylactic doses, UH and EN 40 mg could exert their antithrombotic activity at least partially through a different mechanism of action which involves for instance the vessel endothelial wall binding of similar UH and EN fragments. The results suggest that inhibition of prothrombin consumption may not be clinically relevant for prophylactic treatment with UH. Its relevance in patients receiving LMWH warrants further investigation.
1. SAMAMA, M., BERNARD, P. ,BONNARDOT, J.P., COMBE-TAMZALI, S., LANSON, Y. and TISSOT, E. Low molecular weight hepsrin compared with unfractionated heparin in prevention of post-operative thrombosis. Br. J. Surg. 75,128-131, 1988. 2. PLANES, A., VOCHELLE, N. MAZAS F., MANSAT, C., ZUCMAN, J., LANDAIS, A., PASCARIELLO, J.C., WEILL, D., and BUTEL, J.Prevention of post-operative venous thrombosis : a randomized trial comparing unfractionated heparin with low molecular weight heparin in patients undergoin, ~7total hip replacement. Thromhos. Huemost. 60 (3), 407-410, 1988. 3. TURPIE, A.G.G., LEVINE, M. and HIRSH J. A randomized controlled trial of a low molecular weight heparin (Enoxaparin) to prevent deep vein thrombosis in patients undergoing elective hip surgery. N. Engl. J. Med. 315,925-929, 1986.
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4. SIMONNEAU, G. for the TVPENOX group Study. Subcutaneous fixed dose of enoxaparin (E) versus intravenous adjusted dose of unfractionated heparin (UH) in the treatment of deep venous thrombosis (DVT). Thrombos. Haemost. 65 (6), 754,306a, 1991. 5. HEMKER H.C. The mode of action of heparin in plasma. In : Thrombosis and Haemostasis. Verstraete M., Vermylen J. Lijnen R, Amout N. (Ed). Leuven University 1987, pp. 17-32. 6. SAMAMA, M.M., COMBE, S., HORELLOU, M.H., AUGRAS, D., TRUC, J.B., CONARD, J., GIRARD, P., BARA, L. and POITOUT, PH. Anti-Xa activity and prothrombinase inhibition in patients treated with two different doses of enoxaparin in gynecologic surgery. Thrombos. Res. suppl. XIV, 29-37, 1991. 7. SANDSET, L. and ABILDGAARD, U. Extrinsic pathway inhibitor. The key of blood coagulation initiated by tissue thromboplastin. Haemosrasis 21, 219-239, 1991. 8. SANDSET, L., ABILDGAARD, coagulation pathway inhibitor
U. and LARSEN B.L. Heparin induces release of extrinsic (EPI). Thrombos. Rrs. 50, 803-813, 1988.
9. LINDAHL, A.K., ABILDGAARD, U., LARSEN, B.L., STAALESEN, R., GANGNAESHAMMER, A.K., SANDSET, P.M., NORDFANG, 0. and BECK, T.C. Extrinsic pathway inhibitor (EPI) released to the blood by heparin is a more powerful coagulation inhibitor than recombinant EPI. Thromhos. Res. 62, 607-614, 1991. 10. LINDAHL, A.K., ABILDGAARD, U., LARSEN, B.L., AAMODT, LM., NORDFANG, 0. and BECK, T.C. Extrinsic pathway inhibitor (EPI) and post heparin anticoagulant effect in tissue thromboplasin induced coagulation. Thromhos. Res. suppl. XIV,39-48, 1991. 11. WUN-TZE, Chein. Lipoprotein associated coagulation inhibitor (LACI) is a cofactor for heparin ; synergistic anticoagulant action between LACI and sulfated polysaccharides. Blood 79,430-438, 1992. 12. SCHMIDT, M., BARROWCLIFFE, T.W., GRAY, E., WAITON, G. and HARENBERG, Anti-Xa clotting activities in different hepatic triglyceride lipase preparations from post heparin plasma. Thromhos. Res. 63, 503-508, 199 1. 13. CALLENDER, N.S., RAO, L.V.M., SANDSER, P.M., WARNCRAMER, B.J. and RAPAPORT, S.1. Binding studies of extrinsic pathway inhibitor (EPI) to a cell surface. Blood 76 (suppl. I), 414 a, 1990. 14. NOVOTNY, F. W., PALMIER, M., WUN, T.C., BROZE, J.G. Jr and MILETICH, Purification and properties of heparin releasable lipoprotein associated coagulation inhibitor. Blood 78, 2, 394-400, 1991.
J.P.
15. BROZE, G.J.Jr., WARREN L.A., NOVOTNY, W.F., HIGUCHI, D.A., GIRAR, J.J., MILETICH, J.P. The lipoprotein-associated coagulation inhibitor that inhibit the factor VII-Tissue factor complex, also inhibits factor Xa : insight into its possible mechanism of action. Bloc,d 71, (3), 335-333, 1988. 16. BELL, W.N., ALTON H.G. A brain extract as a substitute for platelet suspensions thromboplastin generation test. Narwe 174, 880-88 1, 1954.
in the
17. BARA, L., BILLAUD, E., GRAMOND, G., KHER, A. and SAMAMA M. Comparative pharmacokinetics of a low molecular weight heparin (PK 10169) and unfractionated heparin after intravenous and subcutaneous administration. Thrombos. Res. 39, 631-636, 1985.
J.
452
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Vol. 69, No. 5
18. CAEN, J., LARRIEU, M.Y. and SAMAMA, M. La consommation de prothrombine. In :
I’Htmostase. Methodes d’exploration et diagnostic pratique (Ed.) L’expansion Scientifique (Paris) deuxieme edition, 1975, pp. 173-176. 19. SANDSET, P.M., ABILDGAARD, U. and PETTERSEN, M. A sensitive assay of extrinsic coagulation pathway inhibitor (EPI in plasma and plasma fractions). Thrombos. Res. 47, 389-400,1987.
20. HEMKER, H.C., WILLEMS, G.M., BEGUIN, S. A computer assisted method to obtain the
prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb. Haemost. 56,9-17, 1986. 21. SAS (Statistical Analysis System) procedure. SAS Institute INC. Box 8000 Cary, North Carolina 275 11, U.S.A. 22. LANE, D.A., DENTON, J., FLYNN, A.M., THUNBERG, L. and LINDAHL, U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem. J. 218,725-732, 1984. 23. LANE, D.A., PEJLER, G., FLYNN, A.M., THOMPSON, E.A. and LINDAHL, U. Neutralization of heparin - related saccharides by histidine-rich glycoprotein and platelet factor 4. J. Biol. Chem. 261,3980-3986, 1986. 24. RUCINSKI, B., NIEWIAROWSKI, S, STRZYZEWSKI, M. HOLT, J.C. and MAYO, K.H. Platelet factor 4 and its C terminal human peptides : heparin binding and clearance from the circulation. Thrombos Haemost. 63, (3), 493-498, 1990. 25. BEGHIN, S., MARDIGUIAN, J., LINDHOUT, T. and HEMKER, H.C. The mode of action
of low molecular weight heparin preparation (PK 10169) and two of its major components on thrombin generation in plasma. Thrombos Haemost. 61 (l), 30-34, 1989. 26. ELLIS, V., SCULLY, M.F. and KAKKAR V.V. The acceleration of the inhibition of prothrombinase complex by heparin. Biochem. J. 233, 161-165, 1986.