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Biomedical subjects

I J Mackie

Publications and source records attributed to I J Mackie.

At least 19 recordsLinked to original sources
PubMed1992

Contact factor proteases and the complexes formed with alpha 2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates.

Plasma from women taking combined oral contraceptives and cold-activated plasma contain proteases which cleave chromogenic substrates in protein C assays in the absence of protein C activators such as Protac. This spontaneous activity makes a background substraction necessary and makes protein C (PC) assays less accurate. We investigated two commonly used substrates < Glu-Pro-Arg-pNA (S-2366) and 2AcOH.H-D-Lys(Cbo)-Pro-Arg-pNA (PC substrate) and found that cold-activated normal and protein C-deficient plasmas gave absorbance values up to 300 times higher than buffer blanks. FXIa cleaves these substrates but activity was not blocked by corn or lima bean trypsin inhibitors, soy bean trypsin inhibitor (SBTI), hirudin or epsilon-amino-n-caproic acid (EACA). Kaolin activation of normal, FXI, FIX, FVIII, FVII and protein C-deficient, but not of FXII or prekallikrein (PKK)-deficient plasmas led to cleavage of chromogenic substrate for protein C. The protein C substrates were cleaved by purified kallikrein and alpha- and beta-FXIIa. Immunoabsorption with alpha 2-macroglobulin (alpha 2M) antibodies removed 60% of the alpha 2M and 70% of the activity on PC Substrate. Gel filtration of normal plasma on Sephadex G-150 gave a single peak of protein C activity and antigen in the included volume. After cold activation of the fractions, a second protein C-like peak appeared in the void volume, but with no detectable protein C antigen. This peak coincided with alpha 2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1992

Some 'antiphospholipid antibodies' bind to beta 2-glycoprotein I in the absence of phospholipid.

Some antiphospholipid antibodies (aPL) only bind to anionic phospholipids in the presence of a serum cofactor, beta 2-glycoprotein I (beta 2GPI). Whether these aPL can bind to beta 2GPI in the absence of phospholipid is controversial. We have purified anticardiolipin antibodies (aCL) from the plasma of four patients and beta 2GPI from normal plasma by solid phase affinity methods. All four aCL bound to cardiolipin and phosphatidylserine in the presence of beta 2GPI but not in its absence. The binding of two of the antibodies to cardiolipin and phosphatidylserine at various concentrations of human beta 2GPI was compared with that obtained using 10% bovine serum. The two antibodies responded differently to increasing beta 2GPI concentrations, and binding to phosphatidylserine was relatively greater than to cardiolipin using human beta 2GPI. All four aCL bound to plastic plates coated with beta 2GPI in the absence of phospholipid, and beta 2GPI in the fluid phase had no effect on binding. Binding to beta 2GPI coated plates was increased equally when bovine serum or bovine albumin were used as the sample diluent in place of gelatine. These findings and those of others have important implications for the design of assays for antiphospholipid antibodies.

Antibodies, Anticardiolipin↗
PubMed1992

The influence of therapeutic blocking of Gp IIb/IIIa on platelet alpha-granular fibrinogen.

Recent evidence suggests that platelet alpha-granule fibrinogen (fg) is derived from the plasma pool. Since platelets from patients with Type I Glanzmann's thrombasthenia (GT) are deficient in intracellular fibrinogen (fg) it was hypothesized that Gp IIb/IIIa could mediate the uptake of fg. To study the potential role of Gp IIb/IIIa in intracellular fg trafficking, the influence of therapeutic blocking of Gp IIb/IIIa on platelet fg was studied in 12 patients with stable ischaemic heart disease. Patients were either given a single intravenous dose of the monoclonal antibody 7E3 Fab (n = 4) or a combination of bolus and continuous infusion up to 24 (n = 3), 36 (n = 3) or 96 h (n = 2). All patients showed grossly prolonged bleeding times with a significant reduction of ex-vivo ADP induced aggregation. Although, surface Gp IIb/IIIa binding sites were consistently reduced in all patients, there was a variable but delayed decrease in platelet fg relative to vWf:Ag in only six out of the 12 patients studied. The reduction in fg appeared dependent upon both dosage and duration of Gp IIb/IIIa blockade. The study provides further evidence for the novel role of Gp IIb/IIIa in the intracellular trafficking of fg to platelet and megakaryocytic alpha-granules.

Adult↗
PubMed1992

Procoagulant changes induced by oral contraceptives are balanced by an increased fibrinolytic tendency.

Oral contraceptives caused increased fibrinogen, FVII, FX, and fibrinolysis. The latter was associated with elevated FXII and PKK, while C1-INH was decreased, ATIII and alpha 2M were unchanged; it could not be accounted for by changes in t-PA, u-PA, PAI, plasminogen, alpha 2-AP, proteins C or S. HCII and alpha 1-PI were increased and may regulate the availability of thrombin and FXIa. The increased FXII/PKK dependent fibrinolytic potential and HCII may offset any increase in thrombin generation, while alpha 1-PI limits intrinsic coagulation.

Adult↗
PubMed1991

Changes in haemostasis after stopping the combined contraceptive pill: implications for major surgery.

OBJECTIVE: To investigate the changes in haemostasis in the three months immediately after stopping the combined contraceptive pill. DESIGN: Prospective randomised study. SETTING: Family planning centre in London. SUBJECTS: 24 women aged 35-45 investigated before, during, and after six months' use of combined oral contraceptives containing 30 micrograms ethinyl oestradiol together with the progestogens desogestrel or gestodene. MAIN OUTCOME MEASURES AND RESULTS: Blood samples were taken immediately before and after six months of oral contraceptive use and one, two, four, six, eight, and 12 weeks after the pill had been stopped. During the six months of oral contraceptive use the plasma concentration of factor X and fibrinogen increased and that of antithrombin III decreased. Between two and six weeks after stopping the pill a rebound phenomenon occurred with plasma concentrations of antithrombin III increasing (mean change from baseline at two weeks 0.06 IU/l and at six weeks 0.10 IU/l) and fibrinogen decreasing (0.26 g/l change at two weeks and 0.40 g/l at six weeks). Factor X concentrations fell gradually and the values at eight weeks were not significantly different from those found before the combined pill was started. CONCLUSION: The combined pill should be stopped at least four weeks before major surgery, which carries the risk of postoperative thrombosis, to allow the potentially prothrombotic haemostatic changes that occur during its use to be corrected.

Adult↗
PubMed1991

Detection of protein C deficiency during oral anticoagulant therapy--use of the protein C:factor VII ratio.

Laboratory diagnosis of protein C deficiency is complicated by the fact that many patients referred for investigation are already being treated with oral anticoagulants. Protein C and factor VII have similar half lives and by using the protein C:factor VII ratio we hoped to be able to detect protein C deficiency in patients receiving oral anticoagulants. We have studied activity levels of protein C and factor VII to produce protein C:factor VII activity ratios in 105 patients receiving oral anticoagulants, 42 normal subjects, and nine patients with known inherited protein C deficiency. The mean ratios for these groups were 1.38, 1.12 and 0.63 respectively. In patients receiving oral anticoagulants, the protein C level showed a poor correlation with the international normalized ratio (INR) value. Reference ranges for protein C at different levels of anticoagulation were therefore considered unsuitable for the identification of inherited protein C deficiency. Five patients with inherited protein C deficiency were studied with and without oral anticoagulation; their protein C:factor VII ratios remained relatively unchanged, despite alterations in the level of the individual proteins. These results suggest that measurement of the protein C:factor VII ratio may help to identify patients with inherited protein C deficiency whilst on oral anticoagulants.

Adolescent↗
PubMed1991

The fibrinolytic response to venous occlusion and the natural anticoagulants in patients with antiphospholipid antibodies both with and without systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) have an increased risk of thrombosis and this is increased in the presence of antiphospholipid antibodies (APA). These APA are also associated with thrombosis in patients who do not have SLE. We compared haemostatic parameters in SLE patients with and without APA, and also compared patients who had APA but not SLE with healthy normal controls. No relationships between the natural anticoagulants, antithrombin III, heparin cofactor II, protein C and protein S, and the presence of APA were found. In the patients with SLE both tissue plasminogen activator antigen and plasminogen activator inhibitor (PAI) were increased, but these changes were not due to APA which had no effect on fibrinolysis in these patients. In the patients with APA who did not have SLE the fibrinolytic response to venous occlusion was reduced due to raised levels of PAI; similar changes have, however, been reported in some patients with idiopathic thrombosis.

Adolescent↗
PubMed1991

Platelet aggregation is inhibited by a nitric oxide-like factor released from human neutrophils in vitro.

Thrombin-induced platelet aggregation was inhibited in vitro by washed human neutrophils. Aggregation was inhibited in a neutrophil concentration dependent manner but glutaraldehyde fixed neutrophils had no significant effect on platelet aggregation. The neutrophil-derived inhibitory factor had the pharmacological profile of nitric oxide. Its action was potentiated by both superoxide dismutase and M&B22, 948, a selective cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase inhibitor. Haemoglobin lessened this inhibitory action of neutrophils. L-Arginine, the substrate for nitric oxide formation, enhanced inhibition, whereas, L-canavanine, a structural analogue of L-arginine, prevented it. Nitric oxide release by neutrophils antagonized platelet ATP secretion and thromboxane B2 release. Inhibition was mediated by nitric oxide activation of guanylate cyclase with a subsequent rise in cyclic GMP. When neutrophils were stimulated with formyl-met-leu-phe, there was a further increase in platelet cyclic GMP. This was enhanced by superoxide dismutase, but lessened by haemoglobin. Leukotriene B4 stimulation of neutrophils promoted inhibition of platelet aggregation. Leukotriene B4 alone had no direct effect on thrombin-induced aggregation of platelets. Platelets, when incubated with neutrophils and stimulated with calcium ionophore A23187, increased leukotriene B4 production by neutrophils in a platelet concentration dependent manner. Platelets alone were unable to release leukotriene B4. The action of platelets in haemostasis is modified as they come into contact with neutrophils. This may be an important physiological mechanism.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Changes in metabolism induced by oral contraceptives containing desogestrel and gestodene in older women.

Forty women aged 35-45 years were investigated to determine changes in haemostasis, lipids and lipoproteins whilst taking combined contraceptive pills containing the new third generation progestogens, desogestrel and gestodene. There was no statistically significant difference between the two preparations in any of the parameters studied. Women taking the combined pill showed increases in fibrinogen and factor X and a reduction in antithrombin III when compared with their control values. There were also small but significant increases in triglycerides and triglyceride-rich lipoproteins. Total high density lipoprotein cholesterol (HDL), high density lipoprotein-2 cholesterol (HDL2), high density lipoprotein-3 cholesterol (HDL3) and apolipoprotein A-1 were all increased at some stages of the treatment cycle, whereas low density lipoprotein cholesterol (LDL) showed a reduction in the first cycle of treatment. The changes in lipids and lipoproteins would not appear to increase the risk of cardiovascular disease, however the effects of the increase in the pro-coagulant factors are uncertain.

Adult↗
PubMed1990

Heparin cofactor II levels are increased by the use of combined oral contraceptives.

Heparin cofactor II (HCII) was assayed by a microtitre amidolytic substrate technique. A linear response was obtained up to 1.5 U/ml and HCII levels were not affected by freezing and thawing the plasma. The assay was validated by comparing HCII and antithrombin III (AT III) levels in AT-III-deficient plasmas and samples from critically ill patients. Higher HCII levels were found in healthy normal women than in healthy normal men (means 1.16 and 0.97 U/ml, respectively, P less than 0.01). A significant increase in HCII levels from 0.86 to 1.10 U/ml (mean values) was seen in healthy normal women starting on combined oral contraceptive (COC) preparations (P less than 0.001). Increased HCII levels were maintained over a 6-month period, but fell towards normal 14 days after stopping COC, although they were still significantly higher than before starting COCs. The discrepancy in HCII level between normal men and women may be due to COC use. In clinical studies, different reference ranges should be used for men and women, and the need for careful questioning about the use of COCs is emphasized.

Adolescent↗
PubMed1989

Normal haemostasis and its regulation.

Regulation of normal haemostasis and blood flow involves complex interactions between plasma proteins and blood cells, including platelets, leukocytes and the endothelial lining of blood vessels. Thrombin acts as a pivot in the maintenance of the haemostatic balance; the vascular endothelial cell in particular limits the generation of thrombin by localisation of anticoagulant processes on its luminal membrane. The endothelial cell synthesises key molecules in this process and also binds exogenously derived molecules, as well as releasing proteins of the fibrinolysis cascade. The thromboresistance of the luminal surface is further regulated by lipoxygenase and cyclo-oxygenase metabolites of unsaturated fatty acids synthesised by the endothelial cell. In response to trauma, inflammatory reactions, normal wound healing and in association with a variety of disease states, the anticoagulant and fibrinolytic mechanisms are downregulated and the procoagulant and thrombotic mechanisms predominate with resultant generation of thrombin, fibrin clot formation and subsequent platelet adhesion and aggregation. Pro-inflammatory and prothrombotic cytokines downregulate the fibrinolytic and activated protein C pathways as well as inducing synthesis of specific procoagulant and prothrombotic mediators by platelets and leukocytes as well as endothelium.

Animals↗
PubMed1989

A plasma inhibitor of platelet aggregation in patients with Lassa fever.

Previous studies have shown that haemorrhage in Lassa fever is associated with abnormal in vitro platelet aggregation and a high mortality. In Sierra Leone we studied platelet aggregation in healthy local subjects, patients with laboratory-confirmed Lassa fever and febrile patients in whom Lassa virus infection was excluded. There were no significant differences in the mean platelet counts of these groups. Patients with fulminant Lassa virus infection showed a gross depression of in-vitro platelet responsiveness to 1 and 5 microM ADP and 4 micrograms/ml collagen compared to other groups (P = 0.0004-0.0008 when compared to healthy controls, P = 0.002-0.0008 when compared to mild Lassa fever patients). When plasma samples from five of these patients were mixed 1:1 with control platelet-rich plasma, a marked inhibition of ADP-induced aggregation was observed. No inhibitory activity was detected in plasma obtained from healthy subjects or febrile control patients. The presence of inhibitor was strongly associated with the occurrence of haemorrhage (P = 0.03), depression of platelet aggregation (P = 0.004) and severity of Lassa fever (P = 0.007).

Adenosine Diphosphate↗
PubMed1989

Familial occurrence of the antiphospholipid syndrome.

In a family of four the whole spectrum of antiphospholipid and associated antibodies was present but without evidence of connective tissue disease. All four members had anticardiolipin antibodies; two had a confirmed lupus anticoagulant. Thrombocytopenia was severe in one and associated with a high titre of antiplatelet antibody, while another member was found to have a positive antiglobulin test. One member also had a low protein C concentration while two had decreased concentration of protein S. Factors that predispose to these antibodies may be environmental as well as genetic. In view of the well known association of spontaneous thrombotic events with some of these antibodies the prognosis for the family members must be guarded.

Autoantibodies↗
PubMed1989

Lupus anticoagulant, anticardiolipin antibodies, and human immunodeficiency virus in haemophilia.

The prevalence of lupus anticoagulant, using the dilute Russell's viper venom time (DRVT), was determined in 22 patients with mild to severe haemophilia A to see if there was any association with the presence of viral disease. Twelve haemophiliacs (58%) were lupus anticoagulant positive, with a mean patient:control ratio of 1.24 (range 1.15-1.52, normal range 0.84-1.06 which partially corrected with lysed, washed platelets). Nine of these patients were IgG or IgM, or both, anticardiolipin antibody positive and nine were human immunodeficiency virus (HIV) antibody positive, but associations between lupus anticoagulant, anticardiolipin antibodies, and HIV antibody positivity were not significant. Mixing studies of normal plasma and immune depleted factor VIII deficient plasma showed that the DRVT ratio increased when the factor VIII concentration fell below 0.15 IU/ml. There was no significant association between plasma factor VIII concentration and positive DRVT results in haemophiliacs. The addition of porcine factor VIII concentrate produced no correction in eight of the 12 with DRVTs indicative of lupus anticoagulant, suggesting that these were prolonged by antiphospholipid activity. It is concluded that the presence of lupus anticoagulant and anticardiolipin antibodies in haemophiliacs may represent an antiphospholipid response to viraemic challenge, not only to HIV but also to other viral antigens, and that a very low factor VIII concentration may produce a false positive DRVT result.

Adolescent↗