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Purpose: The purpose of this study was to compare Pectoralis major myocutaneous flap (PMMC) and Free flaps for reconstruction of post ablative defects in patients undergoing treatment for oral squamous cell carcinoma and to understand the reasons for choosing each from a developing nation perspective., Material and Methods: In the present study, a retrospective study was conducted of the patients treated by either free flaps or PMMC flaps for reconstructive procedures over a five year period in the Department of Oral and Maxillofacial Surgery, Shri Guru Ram Das Institute of Dental Sciences and Research., Results: Out of 90 PMMC flaps, 44 patients (48.8%) had reconstruction due to lack of micro vascular facility at the center, in 39 patients (43.3%) due to financial constraints and in 7 patients (7%) due to associated comorbities. The overall complication rate in PMMC group was 30% as compared to 28% in Free Flap group. Total flap loss was seen in 3 flaps (2 microvascular and 1 PMMC) while marginal necrosis of skin paddle was seen in 12 patients (13%) in the PMMC group., Conclusion: Though, the selection of PMMC flap over free flap was influenced by many factors, results of this study suggest that PMMC flap still has a major role in post-ablative defect reconstruction even in this era of free flaps, especially in developing countries like India., Competing Interests: The authors have no conflicts of interest to declare that are relevant to the content of this article., (© 2022 Craniofacial Research Foundation. Published by Elsevier B.V.)

AP9-cd, a novel lignan composition from Cedrus deodara has significant anticancer potential, and to further enhance its activity, it was lucratively encumbered into solid lipid nanoparticles (SLNs). These nanoparticles were formulated by micro-emulsion technique with 70% drug trap competence. AP9-cd-SLNs were regular, solid, globular particles in the range of 100-200 nm, which were confirmed by electron microscopic studies. Moreover, AP9-cd-SLNs were found to be stable for up to six months in terms of color, particle size, zeta potential, drug content and entrapment. AP9-cd-SLNs have 30-50% higher cytotoxic and apoptotic potential than the AP9-cd alone. The augmented anticancer potential of AP9-cd-SLNs was observed in cytotoxic IC50 value, apoptosis signaling cascade and in Ehrlich ascites tumor (EAT) model. AP9-cd-SLNs induce apoptosis in Molt-4 cells via both intrinsic and extrinsic pathway. Moreover, the dummy nanoparticles (SLNs without AP9-cd) did not have any cytotoxic effect in cancer as well as in normal cells. Consequently, SLNs of AP9-cd significantly augment the apoptotic and antitumor potential of AP9-cd. The present study provides a podium for ornamental the remedial latent via novel delivery systems like solid lipid nanoparticles., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aims: To assess whether immunizing Apob(tm2Sgy)Ldlr(tm1Her)J mice simultaneously with different atherosclerosis-related epitopes engineered in a single recombinant protein is effective in reducing atherosclerotic lesions., Methods and Results: Antigenic epitopes were incorporated into a dendroaspin scaffold: AHC (ApoB100 peptide + hHSP60 peptide [hHSP60(153-163)] + putative epitope derived from Chlamydophila pneumoniae [Cpn]) and AHHC (AHC + hHSP60(303-312)); and were compared with construct A (ApoB peptide), construct H (hHSP60(153-163)), and construct AH (ApoB100 peptide + hHSP60(153-163)). Immunization with 2 multiple-antigenic epitope constructs elicited high levels of antibodies against each epitope in Apob(tm2Sgy)Ldlr(tm1Her)J mice (apart from hHSP60(153-163), which induced a low antibody response). Histological analyses demonstrated that the mice immunized with AHHC and AHC showed significant reductions in the size of atherosclerostic lesions compared with controls (63.8% and 63.2%; P < 0.001, respectively), and significantly greater reductions in lesions size compared with those after immunization with construct A (24.9%; P < 0.01), H (26.8%; P < 0.05), and AH (42.9%; P < 0.001). Moreover, combination of 2 short Cpn peptides along with ApoB and hHSP60 peptides had an additive effect on reducing the lesion without Cpn infection. Reduction in plaque size correlated with cellular infiltration and cytokine/chemokine secretion in serum or by stimulated spleen cells as well as specific cellular immune responses when compared with controls., Conclusions: Immunization of mice with a single construct containing multiple epitopes derived from ApoB100, hHSP60 and Cpn was more effective in reducing early atherosclerotic lesions through the induction of a specific Treg-cell response than was the construct containing either mono- or bi-epitopes. This approach offers attractive opportunities for the design of protein-based, multivalent vaccines against atherosclerosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A simple and sensitive validated LC-MS/MS analytical method was used for determination of curcumin in rat plasma, using nimesulide as internal standard. Analyses were performed on an Agilent LC-MS/MS system using a Chromolith rod™ and isocratic elution with acetonitrile:10 mM ammonium acetate buffer (pH 3.5) (80:20, v/v) at a flow rate of 0.8 ml/min with a total run time of 3 min and an overall recovery of 77.15%. A triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the negative mode was used. Calibration curve in plasma spiked with varying concentration of curcumin were linear over the concentration range of 10-2000 ng/ml with determination coefficient >0.99. The lower limit of quantification was 10 ng/ml. Intra and inter-day variability's (RSD) for extraction of curcumin from plasma were less than 10% and 15% respectively and accuracy was 102.43-108.5%. Multiple reaction monitoring was used to monitor the transition for curcumin (m/z; 367/217 [M-H](-)) and IS (m/z; 307/229). The method was applied for determining curcumin concentration in plasma after peroral administration of 50 mg/kg of free curcumin (C-S) or curcumin loaded solid lipid nanoparticles (C-SLNs) to rats. Results established selectivity and suitability of the method for pharmacokinetic studies of curcumin from C-SLNs., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: HSP60 is emerging as an immunodominant target of autoantibodies in atherosclerosis and recent studies have revealed oxLDL as a key antigen in the development of atherosclerosis. In this study, we assay whether immunizing Apobtm2SgyLdlrtm1Her/J mice with a combination of ApoB and human HSP60 peptides has an additive effect on atheroprotection compared to ApoB or HSP60 peptides applied alone by following atherosclerotic lesion development., Methods and Results: In this study, 2 weeks after the first immunization, Apobtm2SgyLdlrtm1Her/J mice were placed on a high-fat diet for 8 weeks followed by 2 weeks on a normal diet allowing the mice to adapt to the environment before sacrifice. High levels of ApoB and HSP60 antibodies were detectable in week 2 and week 12 following the first immunization with KLH-conjugated ApoB and HSP60 peptides either individually or in combination. Histological analyses demonstrated that mice immunized with both, ApoB and HSP60 peptides, showed the most significant reduction in atherosclerotic lesions (41.3%; p<0.001) compared to a reduction of 14.7% (p<0.05) and 21.1% (p<0.01) in mice immunized with ApoB or HSP60 peptides, respectively; control mice were immunized with either PBS or adjuvant alone. These results were further supported by significant differences in the cellular and humoral immune responses between test animals., Conclusions: Immunization with a combination of ApoB and HSP60 peptide antigens significantly reduced early atherosclerotic lesions in the Apobtm2SgyLdlrtm1Her/J mouse model of atherosclerosis. This approach offers promise as a novel strategy for developing anti-atherosclerotic agents., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: There is not enough clinical evidence to make a strong recommendation on the optimal duration of thromboprophylaxis using low-molecular weight heparins (LMWH) in patients undergoing major cancer surgery., Patients and Methods: CANBESURE is a randomized, double-blind study which enrolled patients admitted for abdominal or pelvic surgery for cancer. They received 3500 IU of bemiparin subcutaneously once daily for 8 days and were then randomized to receive either bemiparin or placebo for 20 additional days. Bilateral venography was performed after 20 days and evaluated blinded. The primary efficacy outcome was the composite of deep vein thrombosis (DVT), non-fatal pulmonary embolism (PE) and all-cause mortality at the end of double-blind period. Major venous thromboembolism (proximal deep-vein thrombosis, non-fatal pulmonary embolism and venous thromboembolism-related deaths) was also evaluated. The primary safety outcome was major bleeding., Results: Six hundred and twenty-five and 488 patients were included in the safety and main efficacy analyzes, respectively. The primary efficacy outcome occurred in 25 out of 248 patients (10.1%) in the bemiparin group and 32 out of 240 (13.3%) in the placebo group (relative risk reduction 24.4%; 95% CI: -23.7-53.8%; P = 0.26). At the end of double-blind period, major venous thromboembolism occurred in 2 (0.8%) and 11 (4.6%) patients, respectively (relative risk reduction 82.4%; 95% CI: 21.5-96.1%; P = 0.010). No significant difference was found in major bleedings., Conclusions: Four weeks compared with 1 week of prophylaxis with bemiparin after abdominal or pelvic cancer surgery did not significantly reduce the primary efficacy outcome, but decreased major venous thromboembolism (VTE) without increasing hemorrhagic complications.

Chronic heart failure (CHF) is traditionally associated with increased risk of thromboembolic complications. Key features of CHF pathophysiology, such as impairment of intracardiac hemodynamics, peripheral blood flow deceleration, neuroendocrine activation, chronic oxidative stress and proinflammatory changes, could explain the predisposition to thromboembolism. However, conclusive epidemiologic data on thromboembolic event incidence in CHF are lacking. Furthermore, the place of antithrombotic therapy in CHF management is still uncertain. Apart from established indications for warfarin (e.g. atrial fibrillation and previous embolic events), there is no robust evidence to support administration of vitamin K antagonists to other patients with CHF, particularly to patients in sinus rhythm. The role of aspirin in preventing thromboembolism in these patients is also controversial. Large randomized trial data on the effectiveness and risks of warfarin and aspirin use in CHF patients with sinus rhythm are forthcoming. This article provides a brief overview of the epidemiologic and pathobiological background of thromboembolism in CHF, and discusses the up-to-date clinical evidence on antithrombotic therapy in detail.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate., (Copyright 2001 Academic Press.)

The benefit of using subcutaneous low molecular weight heparin for the treatment of acute myocardial infarction is not known. The aim of this study was to determine the efficacy of a low molecular weight heparin (dalteparin sodium) for the treatment of acute myocardial infarction in patients not treated with thrombolytic therapy. Twenty-nine cardiological centres from leading hospitals in India participated in this prospective, multicentre, double-blind, placebo-controlled study in two phases which included 1128 patients with acute myocardial infarction. In the acute phase (between day 1 and 3 of admission) all the patients received a weight-adjusted dose of subcutaneous dalteparin (120 IU/kg twice daily). In the second, double-blind phase of acute myocardial infarction, patients were randomised to receive a fixed dose of dalteparin (7,500 IU) or an identical placebo injection for 30 days. A composite primary endpoint of death, reinfarction, recurrence of angina and emergency revascularisation was used. All the 1128 patients with acute myocardial infarction were included in the trial. In the acute phase, the composite primary endpoint was observed in 58 (5.1%) patients. Of 1037 paients who were randomly assigned to receive a fixed dose of dalteparin (n=519) or placebo (n=518), the composite primary event rate was 6.7 percent and 7.0 percent, respectively (RR 0.97; 95% CI 0.62-1.52; p=0.90). To conclude, treatment with dalteparin administered subcutaneously in a weight-adjusted dose of 120 IU/kg twice daily resulted in a lower than expected mortality during the acute phase of myocardial infarction. A lower fixed once daily dose of 7,500 IU during the chronic phase did not confer additional protection.

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

Several chemotaxis methods have been developed which allow the study of different aspects of cell migration. The major limitation of such methods is the lack of a sustained chemotactic signal. Long-term chemotaxis phenomena which are known to take place in vivo have remained largely uninvestigated. Ways to maintain sustained chemotactic signals were sought and the used to investigate the long-term chemotactic effect of platelet-derived growth factor BB (PDGF-BB) on human vascular smooth muscle cells (HVSMC). PDGF-BB was adsorbed onto microcarrier beads and then embedded in agar. PDGF-BB diffusion was slow and a high and sustained local concentration was maintained in the agar. When PDGF-BB-loaded beads embedded in agar were placed at the edge of a tissue culture dish with HVSMC plated in the center, preferential movement was observed in the direction of the PDGF-BB source. This method was subsequently used to study directional movement of HVSMC arising from explants. This report demonstrates that PDGF-BB if present in an anisotropic concentration induces directional cell movement from such explants. By allowing the study of the effect of sustained chemotactic signals upon cultured cells or cells arising from explants, this method may provide a suitable model for investigating in vivo chemotaxis phenomena.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Low-molecular weight heparin has theoretical advantages over aspirin and dipyridamole in maintaining vascular-graft patency by virtue of its better antithrombotic effect and antiproliferative activity on vascular, smooth-muscle cells. We tested the hypothesis that low-molecular weight heparin would be more effective than aspirin and dipyridamole in maintaining graft patency in patients undergoing femoropopliteal bypass grafting. Patients were randomised to receive either a daily injection of 2500 IU low-molecular weight heparin, or 300 mg aspirin with 100 mg dipyridamole 8 hourly for 3 months. 94 patients were randomised to low-molecular weight heparin and 106 to aspirin and dipyridamole. Patients were stratified according to indication for surgery and were followed up for 1 year. Kaplan-Meier estimate of graft patency showed 87% graft survival on low-molecular-weight heparin and 72% on aspirin and dipyridamole at 6 months. At 12 months, the respective figures were 78% and 64%. Stratified survival analysis showed that this benefit was confined to those having salvage surgery (log rank test p = 0.0006); for those having surgery for claudication there was no significant benefit. No major bleeding events occurred in either group. We conclude that low-molecular weight heparin is better than aspirin and dipyridamole in maintaining femoropopliteal-graft patency in patients with critical limb ischaemia undergoing salvage surgery. This treatment should have considerable cost benefits.

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.

Low-molecular-weight heparin (LMWH) is effective in the prevention of postoperative venous thromboembolism but does it have the safety advantages over standard heparin (SH) that have been claimed? In a multicentre randomised trial in 3809 patients undergoing major abdominal surgery (1894 LMWH, 1915 SH) heparin was given preoperatively and continued for at least 5 postoperative days. Patients were assessed in the postoperative period and were followed up for at least 4 weeks, the emphasis being on safety. Major bleeding events occurred in 69 (3.6%) patients in the LMWH group and 91 (4.8%) patients in the SH group (relative risk 0.77, 95% confidence interval 0.56-1.04; p = 0.10). 93 indices of major bleeding were observed in the 69 LMWH patients and 141 in the SH patients. (p = 0.058). Severe bleeding was less frequent in the LMWH group (1.0% vs 1.9%; p = 0.02), as was wound haematoma (1.4% vs 2.7%; p = 0.007). Bleeding episodes with LMWH were less likely to lead to further surgery to evacuate a haematoma or to control bleeding, and injection site bruising was also less common in the LMWH group. No significant differences were found in the efficacy of the two agents. Perioperative death rates were 3.3% in the LMWH group and 2.5% in the SH group; pulmonary emboli were detected in 0.7% and 0.7%; and deep-vein thrombosis was diagnosed in 0.6% of patients in each group. Follow-up was done on 91% of 3699 evaluable patients. There were 19 further deaths (10 LMWH, 9 SH group) and 25 patients with thromboembolic complications (15 and 10). Of the 3 patients with fatal pulmonary emboli during follow-up 2 had received LMWH and 1 SH. The two drugs were of similar efficacy. The primary end point, the frequency of major bleeding, showed a 23% reduction in the LMWH group, but this difference was not significant. The secondary safety end points revealed that LMWH was significantly better than SH. Fatal pulmonary embolism occurs rarely (0.09%) following discharge from hospital so the cost benefit ratio would not justify prolonged prophylaxis in this setting.

Eight unrelated patients with recurrent thromboembolism, a family history of thrombosis, and plasma antithrombin III (ATIII) activity/antigen levels consistent with a diagnosis of heterozygous type I ATIII deficiency were studied by polymerase chain reaction/direct sequencing of ATIII gene exon-coding regions. Frameshift mutations of one base and two bases, respectively, were found to have occurred in two unrelated patients at the same GAG codon (Glu 245) within exon 4 of the ATIII gene. A literature search showed six further hitherto unrecognized deletion "hotspots" in four other human genes. These deletion-prone sites exhibited sufficient sequence homology with each other to derive a consensus sequence (T G A/G A/G G A/C), suggesting that deletion in human genes may not only be non-random but also sequence-directed.

Factor XI deficiency is a rare bleeding diathesis found predominantly in Ashkenazi Jewish kindreds. A recent study of six Jewish patients identified three distinct mutations (Types I, II, and III) in the factor XI gene that were sufficient to fully define the genotypes of the patients. We have investigated 63 patients with factor XI deficiency and find overall allele frequencies of 44% for the type II mutation, 31% for the type III mutation, and 0% for the type I mutation. Therefore, 25% of the mutant factor XI alleles in our sample remain undefined. However, the distribution of mutant alleles is significantly different between Jewish and non-Jewish populations with hitherto undefined mutations accounting for 84% of the disease alleles in non-Jewish patients. Plasma factor XI:C levels were found to differ significantly between different homozygous and compound heterozygous genotypes and the inheritance of the II/III genotype was found to carry an increased risk of the most severe bleeding tendency.

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.

Plasma beta-thromboglobulin (beta-TG), a marker of in-vivo platelet release reaction, was measured in 27 meniscectomy patients, 10 patients who underwent total knee replacements (TKR) (both procedures performed under tourniquet ischaemia), and 10 herniorrhaphy patients. In 22 meniscectomy and 7 TKR patients plasma-thromboxane-B2 (TxB2), a stable end-product of thromboxane-synthetase activity, was also determined. The mean plasma beta-TG and TxB2 had risen significantly within 5 min of release of the tourniquet, indicating increased in-vivo platelet release reaction and prostaglandin synthesis in these patients. In the herniorrhaphy patients beta-TG and TxB2 did not alter significantly postoperatively. In 6 pigs (weighing 20-30 kg) who underwent meniscectomy under tourniquet ischaemia plasma TxB2 rose significantly 15-30 min after release of the tourniquet, and prostacyclin release was significantly lower from veins exposed to ischaemia than from control veins. Large de-endothelialised areas were seen on scanning electron-micrographic sections of affected veins, and these areas were covered with a monolayer of platelets and platelet clumps. These changes associated with tourniquet ischaemia may explain the high incidence of venous thrombosis seen in patients undergoing meniscectomy and TKR.

17 consecutive patients who presented with extensive deep-vein thrombosis, confirmed by phlebography, were included in this study. Of 12 patients who received streptokinase and plasminogen infusion, complete lysis of thrombi was obtained in 8 and partial but extensive lysis in 4. In contrast, thrombi remained unchanged in each of 5 patients who received the same doses of streptokinase infusion only.

We have studied the ability of particulate stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human endothelial cells. Basal release of superoxide (O2-) and hydrogen peroxide from undisturbed monolayers was very low (108 pmol O2- and 75 pmol H2O2 in 3 h from dishes of 3 X 10(5) cells). Addition of 1-micron diameter polystyrene microspheres, which were phagocytosed by the cells progressively, caused a dramatic increase in release of both metabolites; by 3 h, a 13.5- and 6.6-fold increase over controls was observed respectively (P less than 0.001). Addition of formaldehyde-fixed human platelets or chylomicron-size lipid particles also increased production of reactive oxygen species. Similar rises in H2O2 and O2- production were induced by treatment with 10(-7) M phorbol myristate acetate. Pretreatment of endothelial cells with neuraminidase, heparinase or heparitinase to alter their glycocalyx composition substantially enhanced the effect of microspheres on H2O2 and O2- generation. We conclude that the interactions of particles, including platelets and lipids, with endothelial cells leads to the generation of significant pericellular levels of reactive oxygen species. These metabolites can oxidise a wide variety of nearby molecules, leading to cell damage and altered uptake characteristics for lipoproteins containing peroxidized lipids. These effects are exacerbated when endothelial cell glycocalyx composition is disrupted.

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.

The change in blood-viscosity at low shear-rates (0.77 s-1 and 2-62 s-1 was measured in eighteen normal subjects and postoperatively in sixteen patients after administration of 5000 I.U. of subcutaneous heparin. In both groups there was a significant decrease in the mean blood-viscosity 4 to 6 hours after the injection of heparin. This fall in blood-viscosity may be involved in the prophylactic effect of low-dose subcutaneous heparin in preventing venous thrombosis.

A new pulsed multichannel ultrasonic Doppler instrument was used to determine the relative blood-flow in the perforating veins of 16 patients with symptoms attributable to their leg venous systems. 9 patients had leg ulceration and 7 did not. 8 (89%) of the ulcer patients had sustained blood-flow from the deep to the superficial veins ("reverse") during calf muscle contraction, compared to 1 (14%) of the group without leg ulcers. This evidence supports the theory that postphlebitic leg ulceration is the result of retrograde flow of blood in incompetent calf perforating veins during walking.

In a prospective trial, the prophylactic efficacy of low-dose heparin was investigated in 84 consecutive patients undergoing total-hip replacement. 52 of these were randomly allocated to receive heparin or a combination of heparin and dihydroergotamine (D.H.E.); the remaining 32 patients, who were operated on by one orthopaedic team and did not receive prophylaxis, acted as a control group. The frequency of deep-vein thrombosis (D.V.T.), determined by the 125I-fibrinogen test and venography, was 69% in the control group, 32% in the patients receiving heparin alone, and 16% in those receiving heparin and D.H.E. The difference between the control group and each group receiving prophylaxis was statistically significant. The frequency of femoral-vein thrombi, demonstrated by venography, was analysed separately because of its frequent association with major pulmonary embolism. Femoral-vein thrombi developed in 17 (53%) in the control group, while they developed in only 4 patients (8%) in the prophylaxis group (P less than 0.01). 1 patient in the control group died due to massive pulmonary embolism; in another patient, again in the control group, clinical features suggestive of major non-fatal pulmonary embolism developed. This complication was not observed in patients receiving prophylaxis. There was no difference in the amount of operative or postoperative blood-loss in the three groups. In 3 patients in the entire series wound haematomata developed; all 3 were controls and haematomata developed after the administration of streptokinase or large doses of heparin for the treatment of extensive femoral-vein thrombi. By use of a sensitive assay, heparin could be detected in the plasma in 67% of samples in patients in whom D.V.T. did not develop. In contrast, in those patients in whom D.V.T. developed, heparin could be detected in only 21% of blood-samples (P less than 0.01). It is concluded that in patients undergoing total-hip replacement, low-dose heparin prophylaxis is effective in reducing the frequency of life-threatening extensive thrombi in the femoral vein. Prophylaxis using a combination of heparin and D.H.E. requires further evaluation.

The anticoagulant potency in vitro of chondroitin sulphate E has been found to be similar to that of the heparinoids. In purified systems chondroitin sulphate E was shown to be principally an activator of heparin cofactor II. Maximum acceleration of heparin cofactor II:thrombin interaction was 185-fold (9.3 X 10(7) M-1 min-1), antithrombin III:thrombin interaction was 11-fold (4.16 X 10(6) M-1 min-1) and antithrombin III:factor Xa was 146-fold (3.86 X 10(6) M-1 min-1). Chondroitin sulphate E was observed to prolong the thrombin clotting time of fibrinogen in the absence of antithrombin III and heparin cofactor II. The effect appeared to be related to interference in thrombin:fibrinogen interaction rather than in fibrin monomer polymerization.

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.

Deep-vein thrombosis (D.V.T.) was detected by the fibrinogen-uptake test in six out of a total of thirty-one young women undergoing emergency abdominal surgery who gave a history of recent oral contraceptive intake. In contrast, no D.V.T. developed in nineteen similar patients who were not on oral contraceptives (P less than 0-01). Plasma-antithrombin-III activity was significantly lower preoperatively in patients taking oral contraceptives; postoperative D.V.T. subsequently developed in three out of five patients with preoperative antithrombin-III activity below 50%. In seventy-eight dental patients undergoing molar extraction, antithrombin-III activity was measured before, during, and after operation. Activity fell in all patients during operation, but the fall was significantly greater in women taking oral contraceptives (P less than 0-01). The intra-operative fall in antithrombin-III activity was prevented by a small preoperative dose of subcutaneous heparin.

Heparin is localised in mast cells as an activator of the contact system. The potent anticoagulant properties of heparin confine this activation to the top half of the intrinsic pathway, and coagulation is prevented. Certain mast cells contain chondroitin sulphate E, which can also activate the contact system but is a weak inhibitor of coagulation. The localisation of these mast cells to the intestine suggests that coagulation is required for effective inflammatory response in the intestinal wall.

Fatal pulmonary embolism is a major cause of postoperative death. When no prophylaxis is given in general surgery, it has a frequency of about 1%. Apart from the immediate risk to life, one must also consider the late sequelae of extensive deep vein thrombosis--swelling of the legs, varicose veins, ulceration and other trophic changes which represent an equally distressing situation. Recently, three large-scale surveys have been published, where the data of over 100 randomized controlled clinical trials of prophylaxis against venous thromboembolism has been analysed. The purpose of this talk is to provide an overview of the currently available prophylactic methods analysed in these surveys, with an aim to define a policy for preventing venous thromboembolism, with particular emphasis on the surgical patients. There is evidence suggesting that the frequency of fatal pulmonary embolism can be reduced by such prophylactic remedies as low-dose heparin, dextran, the combination of dihydroergotamine plus heparin, and low molecular weight heparin.

A thrombin method for the determination of fibrinogen concentration has been compared with a non-enzymatic sulphite precipitation method on plasma from patients undergoing thrombolytic therapy with an intermittent plasminogen/streptokinase regime. There was very good agreement between the methods even when the determinations were made in the presence of high levels of fibrinogen degradation products (FDP), provided that the thrombin clottable fibrinogen was greater than 1 mg/ml. However, after extensive fibrinogen depletion, when the thrombin method underestimates fibrinogen concentration due to significant clot inhibition by FDP, an overestimate due to co-precipitation of fragments X and Y will be obtained by the sulphite method. This suggests that the simultaneous application of both techniques may provide a simple method of assessing the presence of high levels of anticoagulant FDP in various clinical disorders.

A new technique was elaborated for measuring LDL uptake by rat aortic endothelial cells in vivo, using a fluorescent marker (Dil)-labelled LDL and quantifying the fluorescence in cells selectively removed from the aorta. This technique was used to study the endothelial uptake of LDL modified by activated human monocytes (LDL-A) in comparison with native LDL (LDL-N) protected from oxidation by vitamin E during the preparation. Incubation of LDL with activated monocytes increased endothelial uptake in vivo by 9.3-fold and also by 4.4-fold in cultured confluent porcine endothelium. In contrast, only a 1.5-fold increase in uptake of LDL-A was observed in sparse cultures. Cytotoxicity of monocyte-altered or native LDL did not differ as measured by the [3H]deoxyglucose-release test on cultured endothelium. Our results suggest that modification of LDL in the circulation by monocytes may make an important contribution to atherogenesis.

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.