Trevor W. Barrowcliffe

Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration

Heparin purified by affinity chromatography on anti-thrombin III-Sepharose has been studied by various methods. The specific activities of the materials obtained were in the range 170–230 units/mg as determined by a whole plasma clotting method and 360–780 units/mg as determined by a F.Xa inhibition method. Gel filtration of the material showed that there was a definite molecular size dependency of the specific activities and the activity profiles were markedly different when assay -ed by different methods. These features were also observed (at generally lower activities) with gel filtration fractions of commercial heparin. The possible conclusions regarding the mechanism of heparin anticoagulant action are discussed.

Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Abstract Heparin purified by affinity chromatography on antithrombin III-Sepharose was fractionated according to molecular size by gel filtration. The various fractions obtained were studied with respect to their ability to potentiate the inhibition of thrombin and of Factor Xa in plasma and in a purified system containing antithrombin III. Clotting assays and chromogenic peptide substrate assays were used in parallel. The inhibition of thrombin showed a dependency on the molecular size of the heparin that was similar to that shown earlier for the APTT assay. The inhibition of Factor Xa showed a different dependency, indicating that the mechanisms for heparin-potentiated inhibition of thrombin and Factor Xa by antithrombin III are not entirely the same. The inhibition of thrombin and Factor Xa in plasma differed from that in the pure system and this difference was shown to be dependent on heparin neutralization effects.

Heparin and low-molecular-weight heparin

Heparin is one of the oldest biological medicines, and has an established place in the prevention and treatment of venous thrombosis. Low-molecular-weight heparins (LMWH) have been developed by several manufacturers and have advantages in terms of pharmacokinetics and convenience of administration. They have been shown to be at least as effective and safe as unfractionated heparin and have replaced the latter in many indications. In this article the chemistry, mechanisms of action, measurement of anticoagulant activities, and clinical status of heparin and LMWH are reviewed.

Anticoagulant Activities of High and Low Molecular Weight Heparin Fractions

Summary. The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti‐Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti‐Xa assays, but in APTT assays the LMW fraction had only half the activity of the HMW fraction. Anti‐Xa assays using synthetic substrate S‐222 gave about 20% lower levels than anti‐Xa clotting assays for both heparins. Complete protamine neutralization of the post‐injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti‐Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three in vitro. This non‐neutralizable activity probably accounts for the difference between the anti‐Xa clotting and synthetic substrate assays. Studies by crossed immunoclectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.

Anticoagulant activities of lung and mucous heparins

Abstract It is generally assumed that heparins prepared from different tissues have equivalent actions. However, significant differences were found between lung and mucosal heparins when they were examined in an anti-Factor Xa assay, both in vivo and in vitro . These differences were not seen when the heparins were assayed by an APTT assay. With both types of heparin, anti-Xa activity increased with low molecular weight fractions, while the APTT activity decreased. However, with lung heparin the specific activities by anti-Xa assay were much lower at all molecular weights examined. It is believed that these observations have important implications for the assay and clinical use of heparin.

Inhibition of interleuk:in-2 secretion by factor VIII concentrates: a possible cause of immunosuppression in haemophiliacs

Summary The inhibitory effect of factor VIII concentrate products on IL‐2 secretion by human T‐cells was investigated. The six products used widely in the lJ.K. showed very different activities varying from almost total inhibition to no significant effect. There appeared to be no obvious relationship between inhibitory activity and protein composition but factor VIII itself was not responsible for the effect as affinity purified products were entirely non‐inhibitory. The two wetheated products were most inhibitory whereas dry‐heated products were less inhibitory or non‐inhibitory. However, a wet‐heated version of a non‐inhibitory dry‐heated product was also non‐inhibitory, suggesting that the composition of the concentrate rather than anti‐viral treatment is important for immunosuppressive activity. A product treated by the solvent/detergent procedure showed considerable inhibitory activity. Immunoglobulin and albumin products did not inhibit IL‐2 secretion to any significant extent, but factor IX concentrates were inhibitory. We suggest that inhibition of IL‐2 secretion by factor VIII concentrates may be related to the immunosuppression observed in haemophiliacs treated with high dose factor VIII products and that our results should be considered by clinicians and manufacturers of factor VIII products.

LOW MOLECULAR WEIGHT HEPARIN(S)

It is 10 years since Dr Duncan Thomas’ annotation on heparin, LMW heparin and heparin analogues (Thomas, 1984). At that time, LMW heparin was an experimental new drug being developed by a few companies and only one clinical trial had been published. Since then it has become an established therapeutic material with at least five manufacturers’ products licensed in Europe. and over 50 published clinical trials have demonstrated its therapeutic efficacy. In some clinical indications LMW heparin has replaced unfractionated heparin (UFH) as the drug of choice and the overall turnover is estimated as a thousand million dollars per annum. This annotation will focus on the current clinical status of LMW heparin, and will try to clarify some of the confusion about the differences between LMW and unfractionated heparin and among the various products: in addition, the debate on its mechanism of action and the relative importance of anti-Xa and thrombin inhibition activities will be reviewed.

Discrepancy between one-stage and two-stage assay of factor VIII:C.

Summary. Two methods (one‐stage and two‐stage) are commonly used for the assay of factor VIII clotting activity (VIII:C). We present collected data from seven separate studies of VIII:C assay which show that these methods do not give the same result when comparing concentrates and plasma. On average, two‐stage assays detect 20% more VIII:C activity in concentrates as compared to plasmas than do one‐stage assays.

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4)

Summary. The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti‐Xa and anti‐IIa activities, were measured in the absence and presence of platelet factor 4 (PF4).

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines.

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.