SummaryA low molecular weight heparin (enoxaparin, mean molecular weight ~ 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions.We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chainlength necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways.We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.
We define a standard independent unit (SIU) of heparin as that amount that, in plasma containing 1 μmol of ATIII, raises the (pseudo-)first-order breakdown constant of factor Xa by 1 min-1• These units measure all material with a high affinity for ATIII (HAM); only material above the critical chain length of 17 monosaccharide units (above critical chain length material; ACLM) catalyzes the inactivation of thrombin. An SIU of ACLM is therefore analogously defined as the amount that, in plasma containing 1 μmol of ATIII, will raise the (pseudo-)first-order breakdown constant of thrombin by 1 min-1• Of any given heparin preparation one can determine the specific HAM and ACLM activities in terms of SlU/mg. On the basis of the factor Xa and thrombin breakdown constants found in a plasma sample one can then determine the levels of HAM and ACLM. Preliminary experiments were carried out in plasma samples obtained after subcutaneous injection of unfractionated heparin (UFH) and of two types of low-molecular-weight heparin (LMWH). About three times more of UFH activity than of LMWH activity has to be injected to obtain the same levels of ACLM in the plasma. Only with the LMWHs significant amounts of BCLM are found, which rises higher and persists longer than the ACLM. We determined the course of thrombin generation in platelet-rich plasma (PRP) and in platelet-poor plasma (PPP), as well as in the PPP factor Xa generation curve and the course of prothrombin conversion. The observed inhibitions correlated much better with the levels of ACLM than with those of below critical chain length material. The difference between UFH and LMWHs can therefore not be explained in terms of antithrombin and anti-factor-Xa activity. The essential difference between UFH and LMWH appears in the feedback effect of thrombin in PRP, where thrombin generation is both inhibited and retarded by LMWH, while it is only retarded but hardly inhibited by UFH.
Two preparations of recombinant t-PA obtained from mammalian cells (Boehringer Ingelheim) containing mainly single-chain forms t-PA (sct-PA > 95%) or two-chain forms t-PA (tct-PA > 70%) were compared in vitro for their thrombolytic and fibrinogeno-lytic properties.Thrombolytic activities studied on human hanging clots were similar for both preparations, at plasma concentrations ranging from 62.5 to 1,000 IU/ml. Fibrinogenolysis appeared for both preparations at concentrations higher than 500 IU/ml, after a 90 min incubation at 37°C. For higher concentrations, fibrinogenolysis was dose-dependent, and- there was a trend with sct-PA to provoke less fibrinogenolysis.The turnover of sct-PA was studied in rabbits and cats. After an intravenous bolus injection of 0.2 mg/kg, t-PA activity and antigen showed a double decay disappearance curve. Initial and secondary phases had similar t.1/2 for the activity and the antigen, as well as in both species, which were respectively estimated about 2 min and 20 min.Although the biological and immunological clearance rates were not significantly different, sct-PA antigen values were much higher than the activity.Sct-PA clearance rate was also measured in patients with acute myocardial infarction, treated with intravenous infusion of sct-PA, 100 mg over 3 hours, of which 60 mg during the first hour (n=4). t-PA antigen was regularly measured within 20 min after the end of the first hour infusion.A biphasic disappearance curve was observed, the first phase was extremely rapid with a t.1/2 of 3.2 ± 1.2 min, and the secondary phase had a t.1/2 of 10.4 ± 2.6 min.
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