Francoise Fernandez

A comparison of the thrombolytic and hemorrhagic effects of tissue-type plasminogen activator and streptokinase in rabbits.

Tissue-type plasminogen activator (t-PA) is a promising thrombolytic agent because it can produce thrombolysis without inducing a plasma proteolytic state. It is uncertain if this potentially important feature renders t-PA less hemorrhagic than other plasminogen activators. We have compared the hemorrhagic and thrombolytic effects of t-PA and streptokinase in rabbits. Streptokinase, 4000 U/kg/hr over 4 hr, failed to produce significant thrombolysis and 8000 U/kg/hr streptokinase over 4 hr produced only 28 +/- 6% thrombolysis. Both streptokinase regimens were associated with a plasmin-mediated plasma proteolytic state and both streptokinase regimens produced a significant increase in hemorrhage that was evident within 15 min of beginning the infusion and was progressive over the 4 hr of drug administration. In contrast, t-PA in a dose of 7500 U/kg/hr produced 35 +/- 6% thrombolysis, but it did not produce a plasmin-mediated plasma proteolytic state or a significant increase in hemorrhage over the 4 hr of infusion. t-PA in a dose of 15,000 U/kg/hr produced 85 +/- 4% thrombolysis but was associated with a plasmin-mediated proteolytic state and produced significant bleeding which, in contrast to streptokinase-induced bleeding, was delayed in onset. Therefore, t-PA induced less hemorrhage than streptokinase at doses that produced more effective thrombolysis. Bleeding with both thrombolytic agents was associated with a plasmin-mediated proteolytic state.

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides

Summary. The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HCII) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

The thrombolytic and hemorrhagic effects of tissue type plasminogen activator: Influence of dosage regimens in rabrits

Tissue type plasminogen activator (t-PA) is an effective thrombolytic agent in experimental animals and in humans. We have previously observed that the thrombolytic effect of t-PA persists beyond its time of clearance from the circulation and that wound bleeding induced by a high dose infusion of t-PA is delayed for at least one and a half hours after commencing the infusion. These observations suggest that improved thrombolysis may be obtained with t-PA by infusing a high dose over a short period of time. To test this hypothesis we compared the thrombolytic and hemorrhagic effects of t-PA infused over 240, 60, 30 and 15 minutes in the rabbit. A 4 hour infusion of 30,000 U/kg of t-PA produced 36% thrombolysis. The same dose of t-PA infused over 60, 30 and 15 minutes produced 87%, 88% and 96% thrombolysis, respectively (p less than 0.01). The 1 hour infusion of 30,000 U/kg of t-PA produced a significant increase in blood loss relative to saline infusion (p less than 0.01). The same dose infused over 30 and 15 minutes did not produce significantly more bleeding than saline. A higher dose of t-PA, 60,000 U/kg, infused over 4 hours, produced 95% thrombolysis, but was associated with a significant increase of blood loss (p less than 0.001). Our findings suggest that improved thrombolysis with t-PA with minimal bleeding side-effects is achieved in rabbits when t-PA is administered in a relatively high dose given over a short period of time.

Effects of heparin, its low molecular weight fractions and other glycosaminoglycans on thrombus growth in vivo

Low molecular weight heparin fractions (LMWH) are less hemorrhagic but are as effective as standard heparin (SH) in preventing experimentally-induced venous thrombosis. The effect of LMWH in preventing extension of established thrombi is unknown. We have compared the effects of two LMWHs (CY, PK), and a low molecular weight heparinoid (a dermatan/heparan/chondroitin mixture, OH) with SH on the prevention of extension of established venous thrombi, by measuring their ability to inhibit the accretion of 125I-fibrin onto venous thrombi pre-formed in rabbit jugular veins. Anticoagulant activity was assayed ex vivo by the APTT and a chromogenic anti-Xa assay, and the antithrombotic effect of these glycosaminoglycans was related to their anticoagulant effects. Autologous thrombi were formed in both jugular veins of each rabbit. The rabbits were then injected with 125I-fibrinogen and treated with a bolus dose of glycosaminoglycan or saline, followed by a continuous infusion for 4 hours. All four glycosaminoglycans significantly inhibited 125I-fibrin accretion (p less than 0.001). SH, CY and PK were equipotent at doses of 42.5-62.5 anti-Xa U/kg/hr in preventing fibrin accretion by 50%. Higher doses had no further effect. OH was significantly more potent than the other three glycosaminoglycans at any given dose (p less than 0.005). There was no correlation between the antithrombotic effect and the anticoagulant effects. We conclude that these LMWHs are as effective as SH in preventing extension of established thrombosis.

Abnormal platelet serotonin uptake and binding sites in myeloproliferative disorders.

A simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patients platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patients platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

α2-MACROGLOBULIN AND ANTITHROMBIN III ARE EQUALLY IMPORTANT INHIBITORS OF THROMBIN IN NEONATAL PLASMA

Healthy newborn infants rarely Buffer from thrombosis, despite their low plasma antithrombin III (ATIII) levels. In adults, ATIII is the main inhibitor of thrombin; α2-macroglobulin (α2M) and heparin-cofactor (HCII) are of lesser importance. In infants and children, plasma α2M concentrations are higher than in adults and exceed ATIII activities by about 2-fold. Therefore we determined the contribution of all 3 antiproteases to the inhibition of 125I-thrombin in defibrinated pooled adult and cord plasmas. Thrombin-inhibitor complexes were quantitated by SDS-PAGE. Thrombin was inhibited to a similar extent in both plasmas. The proportions of thrombin complexed to α2M, ATIII and HCII are summarized below:Values are means (SD) of 18 determinations/pooled plasma. The observed differences in distributions are consistent with calculated velocities which favour the complex formation between α2M and thrombin in neonatal plasma. Upon addition of heparin, ATIII became the most important inhibitor of thrombin in cord plasma.Conclusion: In the absence of heparin, α2M and ATIII are equally important inhibitors of thrombin in neonatal plasma. Speculation; High α2M plasma concentrations may protect healthy newborn infants and possibly also ATIII deficient children from thrombosis.