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Biomedical subjects

J H Morrissey

Publications and source records attributed to J H Morrissey.

At least 19 recordsLinked to original sources
PubMed2006

Phospholipid composition controls thromboplastin sensitivity to individual clotting factors.

BACKGROUND: Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. OBJECTIVES: To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. RESULTS: We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin's content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. CONCLUSIONS: Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay.

Adolescent↗
PubMed2006

Traces of factor VIIa modulate thromboplastin sensitivity to factors V, VII, X, and prothrombin.

BACKGROUND: Thromboplastin reagents are used to conduct prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and screen for clotting factor deficiencies. Thromboplastins made from purified, recombinant tissue factor are generally more sensitive to changes in plasma factor (F) VII levels than are thromboplastins prepared from tissue extracts. This may be problematic as FVII's short plasma half-life can result in day-to-day fluctuation during oral anticoagulant therapy. We hypothesized that trace contamination of tissue-derived thromboplastins with FVII(a) blunts sensitivity to plasma FVII levels. METHODS: Traces of purified FVIIa were added to thromboplastin reagents prepared using recombinant human tissue factor and the effect on sensitivity to individual clotting factors was quantified in PT clotting assays. RESULTS AND CONCLUSIONS: Adding 5-100 pm FVIIa not only decreased thromboplastin sensitivity to plasma FVII, it surprisingly increased sensitivity to plasma levels of FV, FX and prothrombin. In addition, traces of FVIIa interacted with changes in the salt content and phospholipid composition of recombinant thromboplastins to further modulate their sensitivities to individual clotting factors. These results help explain how thromboplastin reagents of differing composition exhibit differing sensitivities to individual clotting factor levels. Implications of our results for monitoring oral anticoagulant therapy and other uses of the PT assay are discussed.

Anticoagulants↗
PubMed2004

Rapid and efficient incorporation of tissue factor into liposomes.

Tissue factor (TF), the physiological trigger of the blood clotting cascade, is also the active ingredient in thromboplastin preparations which are widely used in clotting assays such as the prothrombin time (PT) test. A type I integral membrane protein, TF must be incorporated into suitable phospholipid membranes for full procoagulant activity. Several methods exist for incorporating TF into phospholipid vesicles, typically employing the formation of mixed micelles containing detergent, phospholipid and TF, followed by detergent removal or dilution below the critical micelle concentration (CMC). These methods have certain drawbacks: they may take several days to complete, employ expensive detergents, are difficult to scale up, and do not always result in complete detergent removal. In this study we have investigated the use of a variety of detergents [Triton X-100, octaethylene glycol monododecyl ether (C(12)E(8)), cholate, deoxycholate, and n-octyl-beta-D-glucopyranoside], and the use of adsorbent beads (Bio-Beads SM-2) for removing detergent, in processes to incorporate TF into proteoliposomes with high specific activity in coagulation assays. The method we have developed is rapid and readily scalable, yielding thromboplastin preparations with specific activities in plasma clotting assays that are at least as high as those made with detergent dialysis.

Blood Coagulation↗
PubMed2004

Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI).

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.

Humans↗
PubMed2002

Factor VII activation, apolipoprotein A-I and reverse cholesterol transport: possible relevance for postprandial lipaemia.

Postprandial lipaemia is associated with activation of factor VII (FVII) and efflux of cholesterol from tissues to nascent plasma high density lipoproteins (HDL) containing apolipoprotein A-I (apo A-I). To determine whether FVII activation and cholesterol efflux occur together in other situations, the responses to intravenous infusion of HDL-like apo A-I/phosphatidylcholine discs were measured in 10 healthy men. Disc infusion (40 mg apo A-I/kg body weight) over 4 h was followed by increases in HDL cholesteryl ester and plasma apo A-I (p <0.0001). Significant activation of FVII was apparent during infusion in fasting subjects (p = 0.03), activated FVII averaging 123% of baseline value by 12 h (p <0.0001). Plasma thrombin-antithrombin (TAT) complex increased to 156% of baseline level by 12 h (p >0.05) but individual responses differed considerably. Peak TAT post-infusion was associated inversely with peak HDL triglyceride concentration (p = 0.004). The coagulation responses to disc-infusion may be due to transfer of phosphatidylserine to cell surfaces during cholesterol efflux.

Adult↗
PubMed2001

Tissue factor: an enzyme cofactor and a true receptor.

Tissue factor is considered to be the physiologic trigger of the blood clotting system in normal hemostasis and in many--perhaps most--thrombotic diseases. A wealth of new knowledge is available regarding the structure and assembly of the TF:VIIa complex and the role of factor VIIa and tissue factor in hypercoagulable states. The exciting recent finding that tissue factor can function as a signaling receptor, and suggestions that tissue factor may have important, non-hemostatic roles, will be the subject of much additional study in the near future.

Blood Coagulation↗
PubMed2001

Factor V Leiden mutation in one family of Chinese origin.

OBJECTIVE: To investigate the factor V Leiden mutation associated with activated protein C resistance (APCR) in Chinese. METHODS: Thirty "normal" individuals and twenty patients with thrombotic disease from Chinese Han Nationality were studied with APTT +/- APC, PCR followed by MnLI restriction enzyme analysis, PCR based direct sequence-specific primers (PCR-SSP) and DNA sequence analysis. RESULTS: In one healthy control, the activated protein C (APC) sensitivity ratio (SR) was found to be significantly lower (0.8) than that in other normal control (> 2.0). This individual was identified to be heterozygous for FV Leiden mutation (Arg506-Gln). His grand-uncle, father, brother and son were also identified to be heterozygous for FV Leiden. The APC resistance was found in 3 other cases of thrombotic diseases, but with no FV Leiden mutation. CONCLUSION: This is the first four generations family case of FV Leiden mutation associated with APCR reported within Chinese ethnic population. It is note-worthy that more FV Leiden or whether other gene defects may be associated with APC resistance and acquired APCR causing thrombosis in Chinese population.

Activated Protein C Resistance↗
PubMed2000

Comparison of novel hemostatic factors and conventional risk factors for prediction of coronary heart disease.

BACKGROUND: This study sought to assess whether novel markers of hemostatic activity are predictive of coronary heart disease (CHD) and improve risk assessment. METHODS AND RESULTS: Conventional CHD risk factors, the activation peptides of factor IX and factor X, factor VII activity and antigen, activated factor XII, prothrombin fragment 1+2, fibrinopeptide A, and fibrinogen were measured in 1153 men aged 50 to 61 years who were free of myocardial infarction at recruitment. Activated factor VII (VIIa) was measured in 829 men. During 7.8 years of follow-up, 104 had a CHD event. Baseline status was related to outcome by logistic regression by using a modified nested case-control design. Screening performance was judged from receiver operating characteristic curves. A high activated factor XII was associated with increased CHD risk, but low levels were not protective. Plasma VIIa and factor X activation peptide were independently and inversely related to risk. Plasma factor IX activation peptide and fibrinogen were positively associated with risk, but the relations were no longer statistically significant after adjustment for other factors, including VIIa and apoA-I. Other hemostatic markers were not associated with CHD risk. CONCLUSIONS: Hemostatic status did not add significant predictive power to that provided by conventional CHD risk factors yet was able to substitute effectively for these factors.

Biomarkers↗
PubMed2000

Epidemiological and genetic associations of activated factor XII concentration with factor VII activity, fibrinopeptide A concentration, and risk of coronary heart disease in men.

BACKGROUND: The relations of plasma activated factor XII (FXIIa) concentration and a common polymorphism (C46T) of the factor XII gene with hemostatic status and risk of coronary heart disease (CHD) were examined by prospective surveillance. METHODS AND RESULTS: Genotyping for the C46T variant was performed in 2624 men 50 to 61 years of age who were free of CHD at baseline. The genotype distribution was as follows: CC, 56.7%; CT; 36.9%; and TT, 6.6%. Plasma FXIIa was measured by ELISA on 1745 samples collected 1 year after baseline; median levels were (ng/mL) CC, 2.0; CT, 1.4; and TT, 0.8 (P:<0.0001). Respective values for plasma fibrinopeptide A (FPA, nmol/L) were 1.52, 1.35, and 1.15 (P:<0.0001); for factor VII coagulant activity (FVIIc, % standard), 114.5, 116.2, and 109.3 (P:=0.02). Group differences in FVIIc were unchanged by adjustment for body mass index and serum triglycerides. Whereas CHD incidence did not differ significantly by genotype, rates (per 1000 person-years) by thirds of FXIIa distribution were for <1.5 ng/mL, 7. 2; for 1.5 to 2.0 ng/mL, 7.2; and for >2.0 ng/mL, 13.6. Respective hazard ratios with the low third as reference group were 1.01 and 1. 96 (P:=0.007), which were essentially unchanged after allowance for genotype, blood lipids, blood pressure, body mass index, FVIIc, and FPA. CONCLUSIONS: The C46T polymorphism is a determinant of FXIIa, FPA, and possibly FVIIc, suggesting that FXII influences the activity state of the coagulation pathway and FPA cleavage from fibrinogen in vivo. Plasma FXIIa is increased in middle-aged men at high risk of CHD.

Biomarkers↗
PubMed2000

Gender-related differences in thrombogenic factors predicting recurrent cardiac events in patients after acute myocardial infarction. The THROMBO Investigators.

Thrombosis contributes to recurrent coronary events in patients after acute myocardial infarction (AMI), but prognostic significance of thrombogenic factors by gender is unknown. This study aimed to determine gender-related differences in the prognostic significance of thrombogenic factors for predicting cardiac events (nonfatal reinfarction or cardiac death) in postinfarction patients. Blood levels of the following factors were measured 2 months after AMI in 791 men and 254 women: fibrinogen, von Willebrand factor, factor VII and VIIa, plasminogen activator inhibitor, D-dimer, cholesterol, apolipoprotein A-1, apolipoprotein B, lipoprotein(a), triglycerides, and high-density lipoprotein cholesterol. After adjustment for clinical covariates, levels of apolipoprotein A, high-density lipoprotein cholesterol, fibrinogen, and factor VIIa were significantly higher in postinfarction women than men. During a mean 26-month follow-up, there were 67 cardiac events (8.5%) in men and 14 (5.5%) in women (p = 0.11). In the multivariate Cox model, elevated levels of factor VIIa were a significant predictor of cardiac events in women (p = 0.022) but not in men (p = 0.80), with significant gender-related effect (hazard ratio 2.80 vs 0.92, respectively; p <0.05). D-dimer had prognostic value in men (p = 0. 006) but not in women (p = 0.36), although the difference between hazard ratios for men and women was not significant (2.35 vs 1.58, respectively; p = 0.49). In conclusion, elevated levels of factor VIIa are associated with an increased risk of recurrent cardiac events in postinfarction women, but not in men. D-dimer is more predictive for cardiac events in postinfarction men than women. These observations indicate possible gender-related differences in the pathophysiologic mechanisms of recurrent cardiac events.

Apolipoproteins↗
PubMed2000

Influence of fatty acid chain length and cis/trans isomerization on postprandial lipemia and factor VII in healthy subjects (postprandial lipids and factor VII).

Exaggerated postprandial lipemia is believed to be atherogenic and to influence risk of thrombosis. The postprandial effects on plasma triacylglycerol concentration, factor VII coagulant activity (FVII(c)) and activated FVII concentration (FVII(a)) of five high fat meals (5.2 MJ, 90 g fat) enriched with medium triacylglycerols (MCT, 8:0+10:0), palmitate(16:0), stearate (18:0), elaidate(18:1 trans) and oleate(18:1 cis) were compared with those following a low fat meal (5.2 MJ,10 g fat) in 16 healthy subjects using a randomized crossover design. Postprandial lipemia measured as the area under the curve (AUC arbitrary units) for plasma triacylglycerol concentration (mean+/-SE) was greater following the oleate (5.8+/-1. 05), elaidate (4.3+/-0.79) and palmitate (4.1+/-0.64) meals compared with stearate (2.0+/-0.45) and MCT (1.1+/-0.47) meals. Fatty acid analyses of the chylomicron lipids suggested that approximately one fifth of the dietary stearate was not absorbed. FVII(c) increased following the oleate, elaidate and palmitate meals and fell following the low fat meal; the increase in FVII(c) was correlated with the AUC for plasma TAG (r=0.34; P=0.001). FVII(a) concentration increased following all high fat meals but not following the low fat meal. The increase in FVII(a) at 7 h was greater after the oleate meal than after the stearate and MCT meals. These results do not support the hypothesis that dietary stearate and elaidate are responsible for the postprandial increases in FVII associated with high fat intakes.

Adolescent↗
PubMed2000

The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface.

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death.

Antigens, Surface↗
PubMed2000

Haemostatic factors in human peripheral afferent lymph.

Peripheral afferent lymph was obtained by cannulation of a collecting vessel in 17 healthy men (mean age 26 years). Lymph/plasma ratios of all vitamin K-dependent factors were lower than expected from molecular weight. Factor VII, factor IX and tissue factor pathway inhibitor (TFPI) lymph/plasma activity ratios were higher than antigen ratios. Activated factor VII (FVIIa) and TFPI-Xa complex concentrations were higher in lymph than plasma, and the raised FVIIa did not appear to be due to cannulation. The fibrinogen lymph/plasma activity (Clauss) ratio averaged about 20% of the antigen ratio. The result of an ELISA for D-dimer was higher in lymph than plasma, often more than five-fold. This high level in lymph was not explored but may indicate proteolysis of fibrinogen and fibrin with release of D-like and D-dimer-like fragments in interstitial fluid.

Adult↗
PubMed1999

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.

Amino Acid Sequence↗
PubMed1999

Down-regulation of monocyte tissue factor mediated by tissue factor pathway inhibitor and the low density lipoprotein receptor-related protein.

Inflammatory mediators like bacterial lipopolysaccharide induce monocytes to express tissue factor (TF), the cell-surface protein that triggers the blood clotting cascade in hemostasis and thrombotic disease. The physiologic ligand for TF is the serine protease, factor VIIa (FVIIa), and the resulting bimolecular enzyme, TF/FVIIa, can be reversibly inhibited by tissue factor pathway inhibitor (TFPI). Culturing monocytic cells in the presence of both FVIIa and TFPI caused down-regulation of TF expression via reducing its half-life. To exert this effect, FVIIa had to be competent to bind both TF and TFPI, and TFPI had to contain the C-terminal domain required for binding to other cell-surface receptors, including the low density lipoprotein receptor-related protein (LRP). TF down-regulation by FVIIa plus TFPI was abrogated by the 39-kDa receptor-associated protein, which blocks binding of all known ligands to LRP. Furthermore, treatment with FVIIa plus TFPI caused monocyte TF to colocalize with alpha-adaptin, a component of clathrin-coated pits. Thus, in addition to reversibly inhibiting TF/FVIIa catalytic activity, TFPI also mediates the permanent down-regulation of cell-surface TF in monocytic cells via LRP-dependent internalization and degradation. This represents an unusual mechanism for receptor internalization, requiring ligand-dependent bridging of one cell-surface receptor (TF) to a second cell-surface receptor (LRP), the latter being capable of clathrin-mediated internalization.

Cell Line↗
PubMed1999

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C.

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.

Animals↗