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Biomedical subjects

K G Mann

Publications and source records attributed to K G Mann.

At least 19 recordsLinked to original sources
PubMed2006

Atorvastatin and quinapril inhibit blood coagulation in patients with coronary artery disease following 28 days of therapy.

BACKGROUND: We evaluated the antithrombotic effects of statins and angiotensin-converting enzyme inhibitor (ACEI) drugs in patients with coronary artery disease (CAD). METHODS AND RESULTS: Blood coagulation at the site of microvascular injury was assessed in 26 males with CAD before and after treatment with quinapril (10 mg day-1; n=13) or atorvastatin (40 mg day-1; n=13) for 4 weeks and an additional 4 weeks of combined therapy (quinapril+atorvastatin). Rates of prothrombin and factor V activation (FVa), fibrinogen (Fbg) cleavage and FVa inactivation showed that both quinapril and atorvastatin decreased the rates of: formation of thrombin B-chain (by 30.6%, P=0.007; and by 34.3%, P=0.003), formation of thrombin-antithrombin complexes (by 30.4%, P=0.0002; and by 40%, P=0.001), FV activation (by 19.1%, P=0.03; and by 21.8%, P=0.005) and Fbg depletion (by 29.2%, P=0.004; and by 32.7%, P=0.001). Atorvastatin alone accelerated FVa inactivation (P=0.005). A further 4 weeks of combined therapy enhanced most anticoagulant effects only when atorvastatin was added to quinapril. CONCLUSIONS: In CAD patients, atorvastatin and quinapril slowed blood clotting at the site of microvascular injury after 28 days of therapy. Addition of atorvastatin to quinapril, but not quinapril to the statin, enhanced the anticoagulant effects. Our findings might help explain the reduced risk of myocardial infarction or stroke in patients treated with statins and/or ACEIs and the lack of clinical benefits from ACEI added to prior statin therapy in patients at cardiovascular risk.

Aged↗
PubMed2006

Immunologic quantitation of tissue factors.

The large number of conflicting reports on the presence and concentration of circulating tissue factor (TF) in blood generates uncertainties regarding its relevance to hemostasis and association with specific diseases. We believe that the source of these controversies lies in part in the assays used for TF quantitation. We have developed a highly sensitive and specific double monoclonal antibody fluorescence-based immunoassay and integrated it into the Luminex Multi-Analyte Platform. This assay, which uses physiologically relevant standard and appropriate specificity controls, measures TF antigen in recombinant products and natural sources including placenta, plasma, cell lysates and cell membranes. Comparisons of reactivity patterns of various full-length and truncated TFs on an equimolar basis revealed quantitative differences in the immune recognition of TFs by our antibodies in the order of TF 1-263 > 1-242 > 1-218 > placental TF. Despite this differential recognition, all TF species are quantifiable at concentrations < or = 2 pM. Using a calibration curve constructed with recombinant TF 1-263 and plasma from healthy individuals (n = 91), we observed the concentration of TF antigen in plasma to be substantially lower than that generally reported in the literature: TF antigen in plasma of 72 individuals (79%) was below 2 pM (quantitative limit of our assay); TF antigen levels between 2.0 and 5.0 pM could be detected in six individuals (7%); and in 14% (13 plasmas), the non-specific signal was higher than the specific signal, and thus TF levels could not be determined. These differential recognition patterns affect TF quantitation in plasma and should be considered when evaluating plasma TF-like antigen concentrations.

Antibodies, Monoclonal↗
PubMed2006

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Activated Protein C Resistance↗
PubMed2005

The plasma hemostatic proteome: thrombin generation in healthy individuals.

BACKGROUND AND OBJECTIVES: The range of plasma concentrations of hemostatic analytes in the population is wide. In this study these components of blood coagulation phenotype are integrated in an attempt to predict clinical risk. METHODS: We modeled tissue factor (TF)-induced thrombin generation in the control population (N = 473) from the Leiden Thrombophilia Study utilizing a numerical simulation model. Hypothetical thrombin generation curves were established by modeling pro- and anticoagulant factor levels for each individual. These curves were evaluated using parameters which describe the initiation, propagation and termination phases of thrombin generation, i.e. time to 10 nm thrombin (approximate clot time), total thrombin and the maximum rates and levels of thrombin generated. RESULTS AND CONCLUSIONS: The time to 10 nm thrombin varied over a 3-fold range (2.9-9.5 min), maximum levels varied over a approximately 4-fold range (200-800 nm), maximum rates varied approximately 4.8-fold (90-435 nm min(-1)) and total thrombin varied approximately 4.5-fold (39-177 microm s(-1)) within this control population. Thrombin generation curves, defined by the clotting factor concentrations, were distinguished by sex, age, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use (OC > sex > BMI > age). Our results show that the capacity for thrombin generation in response to a TF challenge may represent a method to identify an individual's propensity for developing thrombosis.

Adult↗
PubMed2005

Factor XIII Val34Leu polymorphism and gamma-chain cross-linking at the site of microvascular injury in healthy and coumadin-treated subjects.

Fibrin (Fn) cross-linking by activated factor (F) XIII is essential for clot stability. In vitro, a common Leu34 polymorphism of the FXIIIA-subunit increases the rate of thrombin-mediated FXIII activation, but not cross-linking activity upon complete FXIII activation. The effect of FXIII Val34Leu polymorphism on fibrin(ogen) cross-linking in vivo when vascular injury triggers the blood coagulation has not been studied yet. Using quantitative immunoblotting with antibodies raised against FXIIIA-subunits, fibrinogen, and gamma-gamma-dimers, the rates of FXIIIA cleavage and fibrin(ogen) cross-link formation in the fluid phase of 30-s blood samples collected at the site of microvascular injury were compared in the Leu34-positive and -negative healthy individuals and patients on long-term oral anticoagulation. In addition to accelerated FXIII activation, in healthy subjects the presence of FXIII Leu34 allele was associated with increased soluble gamma-gamma-dimer formation by 40% (1355 +/- 17 microg L(-1) for Leu34 carriers vs. 804.3 +/- 17 microg L(-1) for Leu34 non-carriers; P = 0.028) at the site of microvascular injury. This solution phase effect was abolished in coumadin-treated patients (369.4 +/- 75.9 microg L(-1) for Leu34 carriers vs. 290.5 +/- 35.9 microg L(-1) for Leu34 non-carriers; P > 0.05). The present study indicates that the Leu34 allele affects soluble gamma-gamma-dimer formation in untreated individuals, but not in those receiving acenocoumarol. Our data may help elucidate the impact of the FXIII Val34Leu polymorphism on Fn cross-linking in vivo and its modulation by oral anticoagulants.

Adult↗
PubMed2005

Evaluation of the profile of thrombin generation during the process of whole blood clotting as assessed by thrombelastography.

The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotting in a thrombelastograph (TEG) hemostasis system with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with the eight channels of a TEG system. At different time points, the whole blood TEG reaction cups were kept in a cold quenching solution, centrifuged, and the supernatants were kept at -80 degrees C until assayed for TAT by ELISA. The total thrombus generation (TTG) was calculated from the first derivative of the TEG waveform and was compared with thrombin generation measured by TAT. The two vector values--the TAT thrombin generation data and the corresponding TEG TTG--were analyzed using Pearson correlation coefficients (r) and linear, non-linear and natural log (ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is an exponential curve. Linearizing using the ln of the TAT thrombin generation variable produces the same r (0.94) as of the exponential curve. The prediction equation is y = 8.0465 + 0.0005x (P < or = 0.0001), where y is the TAT thrombin generation variable in the ln transformation and x is the TEG TTG variable. The high magnitude of r and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation by the use of TEG TTG.

Antithrombin III↗
PubMed2005

Thrombin generation profiles in deep venous thrombosis.

BACKGROUND: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. OBJECTIVE: Using an integrated approach that defines an individual's comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. METHODS: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals' comprehensive TF-initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. RESULTS: The greatest risk association was obtained using MaxR; with a 2.6-fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 OR:2.5), BMI (OR:2.9 OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. CONCLUSIONS: Thrombin generation based upon the individual's blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.

Blood Coagulation↗
PubMed2004

Thrombin generation: phenotypic quantitation.

An individual's ability to generate thrombin following tissue factor stimulus was evaluated in 13 healthy male donors in a 6-month study. Thrombin generation in whole blood collected by phlebotomy, contact pathway suppressed by the presence of 100 micro g mL-1 corn trypsin inhibitor, was initiated by the addition of 5 pm tissue factor/10 nm phospholipid. Reactions were quenched at 20 min by the addition of an ethylenediaminetetraacetic acid (EDTA), benzamidine, FPRck cocktail. Thrombin generation was determined by an ELISA for thrombin-antithrombin III (TAT) complex formation. Results showed that the levels of TAT observed varied from 245 to 775 nm. Thrombin production was consistent within each individual, CVi = 11.6%, but varied significantly within the group, CVg = 25.2%, and correlated inversely with an individual's clotting time (r = - 0.54, P = 0.07). No correlations were individually observed between TAT and C-reactive protein, antithrombin III, factors II, V, VII, VIII, IX and X, fibrinogen and prothrombin time. However, computer simulations, which integrated each individual's coagulation factor levels using the Speed Rx method (Hockin et al., J Biol Chem 2002; 277: 18322), predicted maximum active thrombin levels (ranging from calculated values of 220-500 nm) consistent with the empirically determined values. Overall, these data suggest that thrombin generated in whole blood exclusively by tissue factor stimulation can be used as an integrative phenotypic marker to determine an individual's response to a tissue factor challenge.

Adult↗
PubMed2004

Factor VIIa replacement therapy in factor VII deficiency.

Factor (F)VII deficiency is an autosomal recessive disorder for which a replacement therapy is not universally available; recombinant FVIIa has been utilized as a therapeutic substitute. As FVII competes with FVIIa for binding to tissue factor in initiating the extrinsic pathway of blood coagulation, a lower dose of FVIIa replacement in cross-reacting material-negative (CRM-) individuals can achieve hemostasis. Three coagulation models (computational, synthetic and in vitro whole blood) were used to predict the FVIIa levels needed to provide apparent hemostasis in a non-bleeding state. Our whole blood results show that a 'normalized' coagulation profile for FVII-deficient individuals has an initiation phase that ends at 5.8 +/- 0.5 min (clot time) and the propagation phase of thrombin generation (thrombin-antithrombin III) yields a maximum concentration of 380 +/- 29 nmol L(-1). When CRM- FVII-deficient subjects were infused with a prophylactic dose of 23 micro g kg(-1) of recombinant FVIIa, 6-8 h postinfusion resulted in a comparable normalized whole blood profile. This FVIIa concentration (0.3-0.7 nmol L(-1)/equivalent dose: 0.8-1.8 micro g kg(-1)) is approximately 1/10 that currently used in treating FVII-deficient individuals and suggests that therapies should be altered relative to the concentration of the FVII zymogen.

Adolescent↗
PubMed2004

Does the genotype predict the phenotype? Evaluations of the hemostatic proteome.

In this review, the complexity arising from the heterogeneity of the human hemostatic proteome is introduced and discussed with respect to impact on the diagnosis, prophylaxis and therapeutic interventions in thrombotic and hemorrhagic diseases. In the 'healthy' population, coagulation factor levels extend over a 2-4-fold range in concentration. In addition, the qualitative performance of these proteins is governed by many molecular events which are influenced both by genetic instructions which influence post-translational modification and by environmental processes that alter coagulation proteins during circulation. As a consequence, the stimulus-response coupling which follows tissue factor presentation to blood and the subsequent expression of thrombin activity is highly variable even in the 'normal' population. The consequences of this molecular heterogeneity and its potential influence on the diagnosis, prophylaxis and ultimate therapy of coagulation diseases are illustrated. It is the intention of the authors to be provocative; encouraging further investigations to understand the clinical significance of the heterogeneity of the human hemostatic proteome.

Blood Coagulation Factors↗
PubMed2003

Predicting the pharmacology of thrombin inhibitors.

Thrombotic disorders can lead to uncontrolled thrombin generation and clot formation within the circulatory system leading to vascular thrombosis. Direct inhibitors of thrombin have been developed and tested in clinical trials for the treatment of a variety of these thrombotic disorders. The bleeding complications observed during these trials have raised questions about their clinical use. The development of a computer-based model of coagulation using the kinetic rates of individual reactions and concentrations of the constituents involved in each reaction within blood has made it possible to study coagulation pathologies in silico. We present an extension of our initial model of coagulation to include several specific thrombin inhibitors. Using this model we have studied the effect of a variety of inhibitors on thrombin generation and compared these results with the clinically observed data. The data suggest that numerical models will be useful in predicting the effectiveness of inhibitors of coagulation.

Anticoagulants↗
PubMed2003

How factor VIIa works in hemophilia.

The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and "acquired" hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5x normal platelets (10 x 10(8)/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 x 10(8)/mL activated platelets (5x normal); (d) with TF, 10 nM rFVIIa and 3-5x normal nonactivated platelets (6-10 x 10(8)/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.

Blood Platelets↗
PubMed2003

What is all that thrombin for?

The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiation phase, small amounts of factor (F)Xa, FIXa and thrombin are generated. The latter activates the procofactors FV and FVIII to the activated cofactors which together with their companion serine proteases form the intrinsic FX activator (FVIIIa-FIXa) and prothrombinase (FVa-FXa) which generate the bulk of FXa and thrombin during a propagation phase. The clotting process (fibrin formation) occurs at the inception of the propagation phase when only 5-10 nM thrombin has been produced. Consequently, the vast majority (greater than 95%) of thrombin is produced after clotting during the propagation phase of thrombin generation. The blood of individuals with either hemophilia A or hemophilia B has no ability to generate the intrinsic FXase, and hence is unable to support the propagation phase of the reaction. Since clot based assays conclude before the propagation phase they are not sensitive to hemophilia A and B. The inception and magnitude of the propagation phase of thrombin generation is influenced by genetic polymorphisms associated with thrombotic and hemorrhagic disease, by the natural abundance of pro- and anticoagulants in healthy individuals and by pharmacologic interventions which influence thrombotic pathology. Therefore, it is our suspicion that the performance of the entire process of thrombin generation from initiation through propagation and termination phases of the reaction are relevant with respect to both hemorrhagic and thrombotic pathology.

Animals↗
PubMed2003

The function of factor XI in tissue factor-initiated thrombin generation.

The influence of plasma and platelet factor (F)XI on thrombin generation initiated with 10 pm tissue factor (TF) in a synthetic coagulation model was evaluated in the presence of either 2 x 108 mL-1 platelets or the equivalent (2 microm) phospholipids. In either system, with all proteins present at physiological concentrations, FXI (30 nm) had no effect on thrombin generation. With phospholipids in the absence of FXI, an increase in vitamin K-dependent proteins (VKDP) (up to 500%) significantly prolonged the initiation phase of thrombin generation and decreased maximum thrombin levels. The inhibition was principally caused by the elevated prothrombin and FIX concentrations. When 30 nm FXI was added with elevated VKDP and phospholipids, the initiation phase was decreased and the maximum thrombin levels generated substantially increased. In experiments with platelets (with and without plasma FXI), an increase in VKDP had little effect on the initiation phase of thrombin generation. These data indicate that (i) FXI has no effect on thrombin generation at 10 pm TF and physiological concentrations of VKDP; (ii) platelets and plasma FXI are able to compensate for the inhibitory effects of elevated VKDP.

Blood Coagulation↗
PubMed2002

Blood coagulation.

The process of tissue factor initiated blood coagulation is discussed. Reactions of the blood coagulation cascade are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are assembled on a membrane surface in a calcium-dependent manner. These complexes are 105-109-fold more efficient in proteolyses of their natural substrates than enzymes alone. Based upon data acquired using several in vitro models of blood coagulation, tissue factor initiated thrombin generation can be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the generation of nanomolar amounts of thrombin, femto- to picomolar amounts of factors VIIa, IXa, Xa, and XIa, partial activation of platelets, and almost quantitative activation of procofactors, factors V and VIII. The duration of this phase is primarily influenced by concentrations of tissue factor and TFPI. The characteristic features of the propagation phase are: almost quantitative prothrombin activation at a high rate, completion of platelet activation, and solid clot formation. This phase is primarily regulated by antithrombin III and the protein C system. Thrombin generation during the propagation phase is remarkably suppressed in the absence of factor VIII and IX (hemophilia A and B, respectively) and at platelet counts <5% of mean plasma concentration. The majority of data accumulated in in vitro models and discussed in this review are in good agreement with the results of in vivo observations.

Blood Coagulation↗
PubMed2001

Pl(A2) polymorphism of beta(3) integrins is associated with enhanced thrombin generation and impaired antithrombotic action of aspirin at the site of microvascular injury.

BACKGROUND: Mechanisms by which the Pl(A2) (Leu33Pro) polymorphism of beta(3) integrins could lead to an increased risk for coronary events are unclear. This study was designed to examine the effect of this polymorphism on blood coagulation. METHODS AND RESULTS: In normal subjects (12 with Pl(A1A1), 9 with Pl(A1A2), and 3 with Pl(A2A2)), we evaluated the activation of prothrombin, factor V, and factor XIII and fibrinogen removal by quantitative immunoblotting; thrombin-antithrombin III complex generation using ELISA; and levels of fibrinopeptide A and B by high-performance liquid chromatography in blood collected every 30 seconds at sites of standardized microvascular injury before and after 7 days of aspirin ingestion (75 mg/d). Compared with the Pl(A1A1) subjects, the Pl(A2) carriers exhibited higher maximum rates of thrombin B-chain generation (by 31.6%; P=0.005), thrombin-antithrombin III complex generation (by 30.7%; P=0.003), fibrinogen consumption (by 31.3%; P=0.002), prothrombin consumption (by 26.1%; P=0.011), and activation of factor V (by 14.1%; P=0.033) and factor XIII (by 27.0%; P=0.012). In the Pl(A1A1) homozygotes, aspirin ingestion resulted in reductions in the velocity of thrombin B-chain formation (by 32.1%; P=0.007), prothrombin consumption (by 30.4%; P=0.018), factor Va generation (by 28.9%; P=0.014), fibrinogen removal (by 41.2%; P=0.001), and factor XIII activation (by 22.6%; P=0.026). In the Pl(A2) carriers, aspirin did not alter the velocity of all these processes. After aspirin ingestion, fibrinopeptide A and B concentrations in the last 30-second interval were significantly reduced, but only in the Pl(A1A1) subjects. CONCLUSIONS: The presence of the Pl(A2) allele is associated with enhanced thrombin formation and an impaired antithrombotic action of aspirin, which might favor coronary thrombosis in the Pl(A2) carriers.

Adult↗