V.V. Kakkar

Low-molecular-weight heparin and prevention of postoperative deep vein thrombosis.

The efficacy of low-molecular-weight heparin as a prophylactic agent was assessed in 150 consecutive patients over the age of 40 undergoing major abdominal surgery. Fifty of these patients received 1250 activated partial thromboplastin time (APTT) units of low-molecular-weight heparin every 12 hours: three developed isotopic deep vein thrombosis, which was confirmed by phlebography in two cases. The other 100 patients received a single injection of 1850 APTT units of low-molecular-weight heparin. Three of them developed isotopic deep vein thrombosis; phlebography failed to confirm the presence of thrombi in each case. None of the 150 patients studied died from fatal or contributory pulmonary emboli. Low-molecular-weight heparin was not associated with any increase in preoperative or postoperative bleeding. The effect of equal amounts of postoperative bleeding. The effect of equal amounts of low-molecular-weight heparin and unfractionated heparin on the coagulation mechanism during surgery was investigated in another 30 patients. The clotting assays and results of in-vivo platelet function tests indicated that both preparations produced similar effect. Intragroup comparisons, however, showed significant differences in the anti-factor Xa activity, lipoprotein lipase release, and plasma prekallikrein concentrations. A single injection of low-molecular-weight heparin daily is a convenient way of preventing deep vein thrombosis in high-risk patients undergoing major abdominal surgery.

β‐Thromboglobulin, Platelet Production Time and Platelet Function in Vascular Disease

Summary. Plasma β‐thromboglobulin (βTG) levels were measured in 103 healthy controls and 112 patients suffering from either peripheral vascular disease (PVD), or cerebrovascular disease (CVD) or deep vein thrombosis (DVT). Plasma βTG was significantly elevated in 46 PVD patients and 24 recent DVT patients compared to controls, but did not differ significantly in 18 chronic DVT and 24 old CVD patients. In addition, heparin neutralizing activity (HNA) and platelet aggregation induced by adenosine diphosphate, 1‐epinephrine and thrombin were compared in 33 out of the 46 PVD patients to 33 controls. The mean HNA was significantly shorter in the PVD patients than in controls. The rate and extent of platelet aggregation were increased in PVD patients compared to controls, but the difference was not statistically significant. Platelet production time (PPT) was measured in 20 controls, 35 PVD patients, nine chronic DVT and 12 chronic CVD patients; significantly shorter PPT was only observed in 14 patients with advanced PVD compared to controls, suggesting increased platelet consumption in these patients. All four assays (plasma βTG, HNA, platelet aggregation and PPT) were performed in 25 patients; no correlation between the four tests was found in these patients suggesting that the tests were measuring various aspects of platelet function. These results suggest that in vivo platelet consumption as well as platelet aggregation and ‘release reaction’are presumably enhanced in PVD and recent DVT patients and that plasma βTG and PPT assays may be better and more specific indicators of in vivo platelet activation than in vitro platelet aggregation test.

Anticoagulant activities of four unfractionated and fractionated heparins

Abstract Four commercial heparins, two of bovine lung and two of porcine mucosal origin, have been fractionated by gel filtration (and one of them also by ion-exchange chromatography) to yield different fractions whose mean MWs lie in the MW range 7,000–26,000. Three plasma anticoagulant assays have been used to measure the specific activities of the heparin fractions, a kaolin-cephalin clotting time method (KCCT), a calcium-thrombin clotting time method (TCT) and a specific anti-factor Xa assay. The two unfractionated mucosal heparins were found to have higher specific activities when measured in the anti-Xa assay compared to the KCCT assay, while the unfractionated beef lung heparins had lower anti-Xa than KCCT activities. An increase with MW of the heparin fractions was found in specific activities as measured by KCCT and TCT assays over most of the MW range. The low MW heparin fractions (less than 10,500) from all of the heparins had specific activity ratios, KCCT:anti-Xa and TCT:anti-Xa, which were always less than unity. These specific activity ratios increased with increasing MW, usually to greater than unity for the highest MW (greater than 22,000) fractions. There was a tendency for these specific activity ratios to be higher for the beef lung than the mucosal heparin fractions at a given MW. The ability of heparin fractions to inhibit the thrombin induced aggregation of platelets in citrated platelet-rich plasma increased with MW over most of the MW range studied and seemed to follow the specific activities of the fractions when measured by TCT or KCCT assays.

Enhanced in vivo platelet “release reaction” in old healthy individuals

Abstract Plasma β-thromboglobulin (βTG) was measured by radioimmunoassay in 219 healthy subjects age range (12–103), arbitrarily dividied into 3 groups: 127 young, 35 middle aged and 57 old. In 73 of the subjects, plasma platelet factor 4 (PF4) level was also determined by RIA. Plasma βTG (mean 33.9ng/ml), range 3–95) and PF4 (mean 15.2 ng/ml, range 6.62–50) levels increased with age with a significant difference between the mean plasma level of the 3 groups. In addition there was a significant sex difference in old females compared to old males. A significant correlation between the 2 proteins was found in each of the 3 groups of subjects and in the whole group suggesting that both proteins are released in vivo from the same pool and presumably at the same rate. Our results indicate that in vivo platelet activation and “release reaction” is significantly enhanced in old and middle aged subjects compared to young suggesting abnormal hemostatic mechanisms in these individuals. This enhanced platelet activity may reflect a prethrombotic state.

Methods for semi micro or automated determination of thrombin, antithrombin, and heparin cofactor using the substrate, H-d-Phe-Pip-Arg-p-nitroanilide · 2HC1

Methods for the measurement of thrombin and plasma antithrombin, by end point determination at a semi micro level and also by rate assay measurement in a fully automated system have been devised using the thrombin specific chromogenic substrate, H-D-Phe-Pip-Arg-p-nitroanilide. Preliminary defibrination of plasma is avoided in both methods. The semi micro method has been correlated with antitrhombin measured in plasma of postoperative patients by established clotting and immunological assays. The automated method has been found to be highly reproducible and to have less scatter than the other procedures.

Comparison of the effects of low molecular weight heparin and unfractionated heparin on activation of human platelets in vitro

An unfractionated heparin (UFH) and a depolymerised derivative of low molecular weight heparin (LMWH) have been compared for their ability to activate platelets suspended in citrated plasma (PRP) or after washing and suspension in hepes buffered tyrode containing fibrinogen. Neither heparin alone induced aggregation of washed platelets, but UFH and to a much lesser extent LMWH, induced aggregation of platelets in PRP. Both heparins caused significant enhancement of a low concentration of ADP-induced activation of PRP and, again, the effect of LMWH was somewhat less than that of UFH. UFH produced a marked potentiation of ADP-induced activation of washed platelets and LMWH was about a third as potent. In addition, UFH induced a potentiation of PAF-induced aggregation and dense-granule release in PRP, a property not shared by LMWH. In PRP, UFH was three times more potent at inhibiting thrombin-induced aggregation and dense-granule release, as might be expected from their specific activities in the KCCT and thrombin time assay. However, with washed platelets, both heparins were equivalent at inhibiting thrombin-induced aggregation, dense-granule release and elevation of cytosolic free calcium ([Ca++]i) as monitored by quin 2 fluorescence. UFH and LMWH alone did not induce a change in [Ca++]i, nor had they any effect on ADP- or PAF-induced elevation of [Ca++]i.

Selenium enhances prostacyclin production by cultured endothelial cells: Possible explanation for increased bleeding times in volunteers taking selenium as a dietary supplement

Selenium added to the culture medium of confluent pig aortic endothelial cells caused a time-related elevation in the activity of the hydroperoxide scavenging enzyme: glutathione peroxidase. This increased activity was associated with an enhanced ability to produce prostacyclin irregardless of whether the agonist was arachidonic acid or thrombin. Since prostacyclin synthetase is believed to be irreversibly inhibited by alkyl hydroperoxides, we feel that the greater production of prostacyclin by selenium-treated cells as compared with control cells may reflect a protective effect of GSH.Px towards the synthetase enzyme. The results from this study may explain the observations made on a group of human volunteers ingesting selenium as a dietary supplement. After six weeks treatment with selenium, bleeding time in this group was prolonged suggesting an improved ability to synthesize prostacyclin as a result of selenium-dependent glutathione peroxidase activation in the vessel wall.

Molecular weight dependence of the anticoagulant properties of heparin: intravenous and subcutaneous administration of fractionated heparins to man.

Abstract A commercial mucosal heparin preparation has been fractionated by gel filtration into five different MW fractions of approximate mean MWs 11500–21500. These five heparins were randomly administered to 5 normal volunteers by intravenous and subcutaneous injection. The anticoagulant effects of the fractions were then determined by KCCT, anti-Xa clotting and anti-Xa chromogenic substrate assays. Following intravenous injection the high MW fraction produced identical elimination curves in all three assays, but with decreasing MW there was a divergence between results obtained by KCCT and anti-Xa assays. The lowest MW fraction produced between 2–3 times greater heparin levels when measured by anti-Xa assay. This divergence in assay results was produced as a consequence of the differences in specific activities seen when fractionated heparins were assayed in vitro by the different assay methods. That is, low MW heparin fractions had a higher anti-Xa specific activity than that determined by the KCCT assay. Conversely, high MW heparin had a greater specific activity when determined by the KCCT assay. When the response to intravenous heparin injection (measured by KCCT and anti-Xa assays) was compared to the dose administered it was found that the anti-Xa response was greater than expected. This increased plasma heparin like activity was not dependant upon the MW of the heparin fractions, and could not be neutralised by PF 4 . A MW dependance was observed for the absorption of the heparin fractions into the circulation following subcutaneous injection, with low MW heparin producing higher heparin levels determined by all the assays.

Characterisation of a soluble D dimer-E complex in crosslinked fibrin digests

Abstract Further information is presented on the heterogeneity of the fragments released by plasmin digestion of crosslinked human fibrin. Six components (labelled initially 1 to 6) were detected by polyacrylamide gel electrophoresis and the two major components (3 and 4) which make up the D dimer complex were examined in relation to fragments D and E. The more cathodic component of the D dimer complex was shown to consist of E fragments non covalently bound to D dimer fragments, while the other fragment was shown to be D dimer. The lability of the soluble D dimer - E complex to freezing and thawing was observed as was the conversion of D dimer to lower molecular weight fragments. The plasmin induced release of D dimer - E, D dimer, fragments D and E and some very large molecular weight components from crosslinked fibrin should prove useful in understanding fibrin polymerisation and lysis.

A HEPARIN ANALOGUE WITH SPECIFIC ACTION ON ANTITHROMBIN III

The effects of heparin and a semi-synthetic heparin analogue were compared in vivo and in vitro. The two drugs differed strikingly in their in-vitro behaviour: unlike heparin, the heparin analogue had little effect in a specific heparin assay or on overall clotting, as measured by the kaolin-cephalin clotting time (K.C.C.T.). When given by parenteral injection, the heparin analogue had almost the same potentiating effect on antithrombin III as mucous heparin, but without a comparable effect on the K.C.C.T. These observations suggest that the heparin analogue may have desirable characteristics for the prophylaxis of venous thrombosis, since it selectively potentiates antithrombin III in vivo while having little effect on overall clotting.