Michael F. Scully

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.

Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue (TF)-dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be physiological importance in controlling TF-mediated thrombin generation.

Tissue Factor Pathway Inhibitor in Endothelial Cells Colocalizes With Glycolipid Microdomains/Caveolae Regulatory Mechanism(s) of the Anticoagulant Properties of the Endothelium

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

An objective study of alternative methods of heparin administration

Abstract The efficacy and safety of two methods of heparin administration was investigated; 100 patients with calf vein thrombosis were randomly allocated to receive either subcutaneous (SC) or intravenous (IV) heparin for seven days. Venography was performed in each patient to confirm the exact size and site of thrombus and was repeated at the end of heparin treatment. IV heparin was administered using a constant infusion pump. The dose of heparin to be administered was determined by daily estimation of KCCT. In SC group, thrombi increased in size in 2%, remained unchanged in 50% and decreased in 48%. In the IV group, they increased in size in 20%, were unchanged in 62% and decreased in 18%. The difference in decreases in size was significant (p

Acquired Dysfibrinogenaemia in Acute and Chronic Liver Disease

Plasma from patients with both acute and chronic liver disease has been examined for evidence of acquired dysfibrinogenaemia, using electrophoretic methods and coagulation tests. An examination of isolated fibrins upon SDS polyacryamide gel electrophoresis failed to demonstrate any molecular or structural defect associated with the polypeptide chains of the patients’fibrinogen or fibrinogen derivatives produced by thrombin or plasmin. However, purified fibrin monomers isolated from plasma using both Reptilase and thrombin exhibited delayed polymerization rates and the occurrence of acquired dysfibrinogenaemia in liver disease is therefore confirmed.

Substrate-related phosphonopeptides, a new class of thrombin inhibitors

Abstract Novel phosphorus-containing peptidomimetics, D-Aa-Pro-Aa P (OPh) 2 , with lipophilic amino acids at the P 3 positions 1 and amino phosphonic acids with neutral side chains in the P 1 position have been designed and synthesized as thrombin inhibitors.

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.

New peptide boronic acid inhibitors of thrombin

Peptides containing α-amino boronic acids bind to the active site of serine protease, forming highly effective inhibitors. The trigonal boron in these compounds contain a vacant 2p orbital that easily reacts with nucleotides such as the Ser hydroxyl or the His imidazole at the catalytic site of the enzyme to give a tetrahedral transition-state boron adduct as shown in Figure 1, similar to that observed with aldehydes which is stabilised in an analogous manner to the transition state of the substrate, by interactions with secondary binding sites (e.g. oxyanion binding pocket)

Fibronectin in fulminant hepatic failure.

Thirteen out of 17 patients with fulminant hepatic failure had plasma fibronectin concentrations below the normal range (194--472 micrograms/ml), the mean concentration in all 17 patients being 117.9 +/- SE 19.4 micrograms/ml. There was a significant negative correlation between plasma fibronectin concentration and aspartate aminotransferase activity, suggesting that fibronectin is consumed during clearance of hepatocyte debris. The reduced availability of fibronectin may be an important factor in the impaired function of Kuppfer cells in patients with fulminant hepatic failure.

Inhibition of human factor Xa by various plasma protease inhibitors.

Abstract The inhibitory effects of the plasma protease inhibitors antithrombin III, α2-macroglobulin and α1-antitrypsin on the activity of the human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide substrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithrombin III, α2-macroglobulin and α1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, α2-macroglobulin and α1-antitrypsin were 5.8·104, 4.00·104 and 1.36·104 M–1·min–1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as α1-antitrypsin>antithrombin III>;α2-macroglobulin in the ratio 4.64:2.08:1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.