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Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo . We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ± 5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders. [ABSTRACT FROM AUTHOR]

Abstract: Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization. [Copyright &y& Elsevier]

The article focuses on the generation of thrombin in hemorrhage control and vascular occlusion. It mentions the association of common molecular and cellular events with thrombin generation. It indicates that hemorrhage control results in the generation of a stable fibrin-platelet dam. It claims that thrombin functions both as a procoagulant and an anticoagulant.

Abstract: Our research aims to provide quantitatively transparent, biologically realistic descriptions of the processes involved in hemostasis which will permit predictions of the behavior of the coagulation system in normal and pathologic states. We use four models of coagulation: (1) numerical approximations of the tissue factor (Tf) pathway of thrombin generation based upon mechanism and dynamics; (2) Tf activation of the “blood coagulation proteome” from isolated cells and proteins; (3) Tf activated contact pathway inhibited whole blood in vitro; and (4) blood shed from standardized microvascular wounds in vivo. The results from these models are integrated in interactive assessments aimed at achieving convergence of biochemical rigor and biological authenticity. Microvascular injury is the most biologically secure but least accessible to mechanistic study. Numerical models while quantitatively transparent are biologically limited. By the integrated analyses of all four models, we establish observations which require inclusion or discovery of new parameters to achieve mechanistically interpretable biological reality. Discoveries made in this fashion have included thrombin''s role in the initiation phase, TFPI/ATIII/APC synergy interactions, rfVIIa in fVII deficiency, the roles of fVIII and fIX in the Tf reaction, and the cleavage of fIX by fXa membrane. Ideally, our results will provide descriptions which predict the behavior of the biological blood coagulation system under normal and pathologic conditions. [Copyright &y& Elsevier]

Expression and purification of proteins in recombinant DNA systems is a powerful and widely used technique. Frequently there is the need to express the protein of interest as a fusion protein or chimeric protein. Fusion protein technology is frequently used to attach a “signal” which can be used for subsequent localization of the protein or a “carrier” which can be used to deliver a “therapeutic” such as a radioactive molecule to a specific site. In addition to these applications, fusion protein technology can be employed for several other useful purposes. Of these, the most frequent reason is to provide a ‘tag’ or ‘handle’ which will aid in the purification of the protein. Another useful purpose is to improve the expression or folding of the protein of interest. In these latter two situations, it is often necessary to remove the fusion partner before the recombinant protein of interest can be used for further studies. This removal process involves the insertion of a unique amino acid sequence that is susceptible to cleavage by a highly specific protease. Thrombin and factor Xa are the most frequently used proteases for this application. The purpose of this review is to discuss the application of thrombin and factor Xa for the cleavage of fusion proteins. It is emphasized that while these enzymes are quite specific for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest. It is necessary to characterize the protein of interest after cleavage from the affinity label to assure that there are no changes in the covalent structure of the protein of interest. Examples are presented which describe the proteolysis of the protein of interest by either factor Xa or thrombin. [Copyright &y& Elsevier]

Provides a brief summary of the evolution of knowledge with respect to present-day concepts of thrombin generation via the tissue factor pathway and its regulation. Antithrombin III; Vitamin K-dependent proteins; Regulation of the hemostatic process; Production of thrombin by a series of proteolytic events.

Reports on a study that evaluated the activation of a blood clotting chemical called human factor VII by a variety of potential activators in the presence and absence of mixed phospholipid vesicles. Factor VIIa-tissue factor complex formation; Evaluation of initial factor VII activation rates; Kinetic constants for factor VII activation.

Introduces the articles on the physiological importance of thrombin featured in the September 2003 supplement issue of the 'Chest' journal. Contribution of thrombin to activation of factor VII, V, VII, XI and XIII; Regulation and functions of thrombin; Contribution of thrombin to platelet activation.

Presents a letter to the editor about plasma clotting activity of blood tissue factor.

Abstract: Tissue factor plays a primary role in both hemorrhage control and thrombosis depending upon whether its presentation is extravascular or intravascular. The molecular architecture and function of the tissue factor molecule and its role in the activations of factor IX and factor X have been elegantly elucidated but controversies prevail with respect to distinctions between tissue factor sources and tissue factor “activity.” This presentation will review data on the architecture and functions of the tissue factor-factor VIIa complex and discuss the elements of the controversies associated with tissue factor presentation in both normal and pathologic milieu. [Copyright &y& Elsevier]

A letter to the editor is presented in response to the article “Overbuilding Research Capacity,” by B. Alberts in the September 10, 2010 issue.

The generation of the enzyme thrombin from its precursor prothrombin is the central event of the blood coagulation process, which is essential to hemostasis and the culprit in thrombosis. Thrombin is produced by a complex series of proteolytic events that are initiated when cryptic tissue factor interacts with plasma factor VIIa to initiate the complex series of events leading to the formation of the blood coagulation enzyme complexes that lead to the efficient generation of the enzyme. During these processes, thrombin contributes to both the generation of the catalysts involved in its ultimate production and to the catalysts that lead to attenuation of its production. Thrombin-catalyzed events both enhance and diminish the process of thrombin generation, which is down-regulated by stoichiometric and dynamic inhibitory processes. The combinations of intensities of activation and inhibition processes provide tight regulation of the hemostatic process, establishing reaction thresholds, essentially leading to an "on/off" switch. This review provides a brief summary of the evolution of knowledge with respect to present-day concepts of thrombin generation via the tissue factor pathway and its regulation. [ABSTRACT FROM AUTHOR]

Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann–Whitney U test, and logistic regression. Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = −0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical. [ABSTRACT FROM AUTHOR]

This study investigates the dynamics of zymogen activation when both extrinsic tenase and prothrombinase are assembled on an appropriate membrane. Although the activation of prothrombin by surface-localized prothrombinase is clearly mediated by flow-induced dilutional effects, we find that when factor X is activated in isolation by surface-localized extrinsic tenase, it exhibits characteristics of diffusion-mediated activation in which diffusion of substrate to the catalytically active region is rate-limiting. When prothrombin and factor X are activated coincident with each other, competition for available membrane binding sites masks the diffusion-limiting effects of factor X activation. To verify the role of membrane binding in the activation of factor X by extrinsic tenase under flow conditions, we demonstrate that bovine lactadherin competes for both factor X and Xa binding sites, limiting factor X activation and forcing the release of bound factor Xa from the membrane at a venous shear rate (100 s-1). Finally, we present steady-state models of prothrombin and factor X activation under flow showing that zymogen and enzyme membrane binding events further regulate the coagulation process in an open system representative of the vasculature geometry. [ABSTRACT FROM AUTHOR]

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TFfactor Vila (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (Vmax) of the complex increased in the order rTF1-243 (Escherichia coli) < rTF1-263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the Km of FX for rTF1-263-FVIIa remained unchanged after deglycosylation, whereas the kcat decreased slightly. A pronounced decrease, 4-fold, in kcat was observed for pTFFVIIa upon deglycosylation, whereas the Km was minimally altered. The parameters of FX activation by both rTF1-263D-FVIIa and pTFD-FVIIa were identical and similar to those for rTF1-243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF1-263 and pTF. The carbohydrates of rTF1-263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars. [ABSTRACT FROM AUTHOR]

Deterministic mathematical models of biochemical processes operate as if the empirically derived rate constants governing the dynamics are known with certainty. Our objective in this study was to explore the sensitivity of a deterministic model of blood coagulation to variations in the values of its 44 rate constants. This was accomplished for each rate constant at a given time by defining a normalized ensemble standard deviation (wkif(t)) that accounted for the sensitivity of the predicted concentration of each protein species to variation in that rate constant (from 10 to 1000% of the accepted value). A mean coefficient of variation derived from (wkif(t)) values for all protein species was defined to quantify the overall variation introduced into the model's predictive capacity at that time by the assumed uncertainty in that rate constant. A time-average value of the coefficient of variation over the 20-min simulation for each rate constant was then used to rank rate constants. The model's predictive capacity is particularly sensitive (50% of the aggregate variation) to uncertainty in five rate constants involved in the regulation of the formation and function of the factor VIIa–tissue factor complex. Therefore, our analysis has identified specific rate constants to which the predictive capability of this model is most sensitive and thus where improvements in measurement accuracy will yield the greatest increase in predictive capability. [ABSTRACT FROM PUBLISHER]

Studies focused on patient responsiveness to antiplatelet therapies, particularly aspirin and clopidogrel, have increased in recent years. However, the relations of in vivo platelet function and adverse clinical events to results of ex vivo platelet function tests remain largely unknown. This article describes current methods of measuring platelet function in various clinical and research situations and their advantages and disadvantages, reviews evidence for antiplatelet response variability and resistance, discusses the potential pitfalls of monitoring platelet function, and demonstrates emerging data supporting the positive clinical and treatment implications of platelet function testing. [Copyright &y& Elsevier]

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. structurally, lithe is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1-A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors. [ABSTRACT FROM AUTHOR]

Thrombin activated factor Va (factor V[sub IIa], residues 1-709 and 1546-2196) has an apparent dissociation constant (K[sub d,app]) for factor Xa within prothrombinase of ∼0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp[sup 697], Asp[sup 1509], and Asp[sup 1514] to produce a molecule (factor V[sub NN]) that is composed of a M[sub r] 100,000 heavy chain (amino acid residues 1-696) and a M[sub r] 80,000 light chain (amino acid residues 1509/1514-2196). Factor V[sub NN], has a K[sub d,app] for factor Xa of 4 nM and reduced clotting activity. Cleavage of factor V[sub IIa] by NN at Asp[sup 697] results in a cofactor that loses ∼60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg[sup 1018] and Arg[sup 1545] to produce a M[sub r] 150,000 heavy chain and M[sub r] 74,000 light chain (factor V[sub RVV], residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor V[sub NN] at Arg[sup 1545] by α-thrombin (factor V[sub NN/IIa]) or RVV (factor V[sub NN/RVV]) leads to enhanced affinity of the cofactor for factor Xa (K[sub d,app] ∼ 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg[sup 1545] and formation of the light chain of factor V[sub IIa] is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor. [ABSTRACT FROM AUTHOR]

The initiation of many physiologically important biochemical reactions is dependent on the binding of a molecule or molecules from solution to an appropriate solid surface such as a cell membrane. The quantitative study of dynamic interactions of molecules in solution with immobilized surfaces under physiologically relevant conditions presents a significant physical problem. Traditionally these measurements have been performed by discontinuous, nonequilibrium assays that cannot accurately model in vivo reaction mechanisms. Total internal reflection fluorescence spectroscopy (TIRFS) is an analytical technique that is based on the excitation of fluorescence by means of an exponentially decaying surface energy wave called the evanescent wave. The limited ‘‘penetration depth’’ of the evanescent wave leads to excitation of fluorescent molecules at or near the surface. This phenomena makes TIRFS uniquely qualified for the dynamic fluorescence study of liquid-solid interface binding reactions. The instrument described here permits the powerful technique of TIRFS to be used by biomedical researchers in their investigation of biochemical interface reactions. The fully automated, menu driven instrument allows the real time dynamic measurement of binding of fluorescent-labeled molecules in solution at physiologically relevant concentrations to appropriate immobilized surfaces.The novel design of the total internal reflection element enables the study of binding from essentially opaque and highly scattering solutions such as whole blood. Measurements may be performed under static, stirred, or flow conditions. The presently described TIRFS instrument is capable of tracking a binding reaction with evanescent fields established by two identical reflecting light beams with different reflection angles. This instrumental feature allows the dynamic measurement not only of the primary binding event but also of fluctuations in the distance between the... [ABSTRACT FROM AUTHOR]

Abstract We previously showed that personalizing prophylaxis on the basis of an individual’s pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis. A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains. Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing. Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis. [ABSTRACT FROM AUTHOR]

Background: Beyond localized damage to the circulatory system and surrounding tissue, trauma stresses endothelial cells throughout the vasculature, potentially leading to hemorrhagic or thrombotic complications away from the injury site.Objective: Use a whole blood endothelial cell model to define the effects of crystalloid fluid therapy on protein C pathway regulation of tissue factor-initiated coagulation.Methods: Tissue factor-initiated coagulation was studied in the presence of EA.hy926 cells. Blood was diluted to 70% or 40% using normal saline or lactated ringers. Analyses of coagulation dynamics included clot times, thrombin formation (thrombin-antithrombin complex), FV activation/inactivation, fibrinogen consumption, FXIII activation, and platelet activation.Results: In all donors, the onset of thrombin generation was not altered in 70% blood using either diluent; with the blood component reduced to 40%, clot time was prolonged two-fold when normal saline was utilized but was unchanged with lactated ringers. The timing of the activations of FV, fibrinogen, and platelets paralleled the effects of dilution on clot times. Extensive inactivation of FVa was observed in undiluted blood and where lactated ringers was the diluent but not in trials with 40% blood/60% normal saline.Conclusion: Feedback inhibition of tissue factor-initiated coagulation by the protein C pathway is not compromised by hemodilution with crystalloids. [ABSTRACT FROM AUTHOR]

Introduction: An elevated procoagulant activity observed in trauma patients is, in part, related to tissue factor (TF) located on blood cells and microparticles. However, analysis of trauma patient plasma indicates that there are other contributor(s) to the procoagulant activity. We hypothesize that factor (F)XIa and FIXa are responsible for an additional procoagulant activity in burn patients.Methods: Multiple time-point plasma samples from 56 burn patients (total number of samples was 471; up to 20 time-points/patient collected in 3 weeks following admission) were evaluated in a thrombin generation assay using inhibitory antibodies to TF, FIXa and FXIa.Results: Due to the limited volume of some samples, not all were analyzed for all three proteins. At admission, 10 of 53 patients (19%) had active TF, 53 of 55 (96%) had FXIa and 48 of 55 (87%) had FIXa in their plasma. 34 patients of 56 enrolled (61%) showed TF activity at one or more time-points. All patients had FXIa and 96% had FIXa at one or more time-points. Overall, TF was observed in 99 of 455 samples analyzed (22%), FXIa in 424 of 471 (90%) and FIXa in 244 of 471 (52%). The concentration of TF was relatively low and varied between 0 and 2.1pM, whereas that of FXIa was higher, exceeding 100pM in some samples. The majority of samples with FIXa had it at sub-nanomolar concentrations. No TF, FXIa and FIXa activity was detected in plasma from healthy individuals.Conclusions: For the first time reported, the majority of plasma samples from burn patients have active FXIa and FIXa, with a significant fraction of them having active TF. The concentration of all three proteins varies in a wide range. [ABSTRACT FROM AUTHOR]

Background Studies associating coagulopathic changes with burn injury have relied on limited tests such as partial thromboplastin time (PTT) and international normalized ratio (INR). Understanding the clotting dynamics and associated risk factors after burn injury could influence management. This work aimed to identify real-time changes in coagulation after burn injury not indicated by PTT or INR alone. Materials and methods Nine burn-injured patients at a regional burn center were enrolled for blood collection at admission and set intervals over 96 h. Patient demographics, management, and laboratory data (PTT and INR) were collected. Plasma assays determined factors II, V, VII, VIII, IX, X, XI, antithrombin, and protein C functional activity as well as PAP, D-Dimer, fibrin monomer, TFPI, IL-1b, IL-6, IL-10, IL-12p.70, and TNF-α concentrations. Results Overall, five patients died. These patients had higher mortality scores and were more acidotic. All patients had normal coagulation studies (INR < 1.5, PTT < 45 s) within 24 h of admission, and only two were abnormal after. Increased factor VIII and IX activity were identified in seven patients at admission. Decreased antithrombin and protein C activity were seen in all patients. Patients had increased PAP, D-Dimer, and fibrin monomer concentrations throughout their hospital course. At admission, increased fold changes were seen in IL-6 (2.5–117) and IL-10 (2.4–32), whereas IL-1b and TNF-α levels were depressed in all patients. Conclusions Extensive changes not identified by PTT or INR were seen after burn injury that may explain perturbed coagulation in these patients. This approach further characterizes the impact thermal injury has on coagulation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms. Methods: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results. Results: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography. Conclusion and significance: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF. [Copyright &y& Elsevier]

Abstract: Background: Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties. Methods: The status of disulfides in recombinant TF1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function. Results: In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function. Conclusion: The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis. General significance: Results of this study advance our knowledge on TF structure/function relationships. [Copyright &y& Elsevier]

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice. [ABSTRACT FROM AUTHOR]

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anticoagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released. [ABSTRACT FROM AUTHOR]

Prothrombin activation can proceed through the intermediates melzothrom-bin or prethrombin-2. To assess the con-tributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)-activated blood, immunoassays were developed that measure the meizothrom-bin antithrombin (mTAT) and a-thrombin antithrombln (aTAT) complexes. We deter-mined that Tf-actlvated blood produced both aTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn tryp-sin inhibitor-treated blood was fraction-ated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initia-tion phase and a marked decrease in the rate and extent of aTAT formation. Only the addition of RBCs to PRP was capable of normalizing aTAT generation. FACS on glycophorin A-positive cells showed that approximately 0.6% of the RBC popula-tion expresses phosphatidylserlne and binds prothrombinase (FITC Xa-factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that sup-ports prothrombin activation through the meizothrombin pathway. [ABSTRACT FROM AUTHOR]

Information about the convention of the National Heart, Lung and Blood Institute (NHLBI) working group is presented. The working group brought together basic scientists, genetic epidemiologists, clinical trialists, interventionalists and cardiologists. The working group discussed challenges for therapy in guided antithrombotic in domains such as the roles of stakeholders, pharmacogenetics and pharmacology, antithrombotic metrics and therapeutic strategies.

The underlying cause of thrombosis in a large protein C (PC) deficient Vermont kindred appears to be multicausal and not explained by PC deficiency alone. We evaluated the contribution of coagulation factors to thrombin generation in this population utilizing a mathematical model that incorporates a mechanistic description of the PC pathway. Thrombin generation profiles for each individual were generated with and without the contribution of the PC pathway. Parameters that describe thrombin generation: maximum level (MaxL) and rate (MaxR), their respective times (TMaxL, TMaxR), area under the curve (AUC) and clotting time (CT) were examined in individuals ±PC mutation, ±prothrombin G20210A polymorphism and ±thrombosis history (DVT or PE). This family (n = 364) is shifted towards greater thrombin generation relative to the mean physiologic control. When this family was analyzed with the PC pathway, our results showed that: carriers of the PC mutation (n = 81) had higher MaxL and MaxR and greater AUC (all p<0.001) than non-carriers (n = 283); and individuals with a DVT and/or PE history (n = 13) had higher MaxL (p = 0.005) and greater AUC (p<0.001) than individuals without a thrombosis history (n = 351). These differences were further stratified by gender, with women in all categories generating more thrombin than males. These results show that all individuals within this family with or without PC deficiency have an increased baseline procoagulant potential reflective of increased thrombin generation. In addition, variations within the plasma composition of each individual can further segregate out increased procoagulant phenotypes, with gender-associated plasma compositional differences playing a large role. [ABSTRACT FROM AUTHOR]

Eur J Clin Invest 2011 Abstract Background Elevated factor (F)XI and tissue factor (TF) have been reported to occur in patients with acute ischemic stroke (AIS). We sought to investigate whether circulating activated FXI (FXIa) and TF on admission can predict clinical outcomes in patients with acute cerebrovascular events. Materials and methods In the observational study, we evaluated 205 consecutive patients aged 70 years or less within the first 72 h of acute event, including 140 with AIS and 65 with transient ischemic attack (TIA). Plasma TF and FXIa activity were determined on admission in clotting assays by measuring the response to inhibitory monoclonal antibodies. Results Active TF and FXIa activity were detected in 58 (28·9%) and 132 (64·4%) patients on admission, respectively. Active TF was detected in 45 of the 136 AIS patients with available TF levels (33·1%) and 13 of the 65 patients with acute TIA (20%; P = 0·05). Corresponding values for FXIa were 99 of the 140 (70·7%) and 33 of the 65 (50·8%; P = 0·006), respectively. Patients with detectable TF were more frequently women and hypertensive, while subjects with detectable FXIa had more often diabetes and higher levels of fibrinogen, C-reactive protein and interleukin-6 (all P < 0·05). Patients with detectable FXIa but not TF had higher National Institutes of Health Stroke Scale score, higher modified Rankin scale score and lower Barthel Index at discharge (all P < 0·05). Conclusions Circulating active TF and FXIa occur frequently in acute cerebrovascular ischemic events. Active FXIa in plasma might be useful as a novel risk marker of worse functional outcomes in patients with acute cerebrovascular events. [ABSTRACT FROM AUTHOR]

In terms of its soluble precursors, the coagulation proteome varies quantitatively among apparently healthy individuals. The significance of this variability remains obscure, in part because it is the backdrop against which the hemostatic consequences of more dramatic composition differences are studied. In this study we have defined the consequences of normal range variation of components of the coagulation proteome by using a mechanism-based computational approach that translates coagulation factor concentration data into a representation of an individual's thrombin generation potential. A novel graphical method is used to integrate standard measures that characterize thrombin generation in both empirical and computational models (e.g max rate, max level, total thrombin, time to 2 nM thrombin ("clot time")) to visualize how normal range variation in coagulation factors results in unique thrombin generation phenotypes. Unique ensembles of the 8 coagulation factors encompassing the limits of normal range variation were used as initial conditions for the computational modeling, each ensemble representing "an individual" in a theoretical healthy population. These "individuals" with unremarkable proteome composition was then compared to actual normal and "abnormal" individuals, i.e. factor ensembles measured in apparently healthy individuals, actual coagulopathic individuals or artificially constructed factor ensembles representing individuals with specific factor deficiencies. A sensitivity analysis was performed to rank either individual factors or all possible pairs of factors in terms of their contribution to the overall distribution of thrombin generation phenotypes. Key findings of these analyses include: normal range variation of coagulation factors yields thrombin generation phenotypes indistinguishable from individuals with some, but not all, coagulopathies examined; coordinate variation of certain pairs of factors within their normal ranges disproportionately results in extreme thrombin generation phenotypes, implying that measurement of a smaller set of factors may be sufficient to identify individuals with aberrant thrombin generation potential despite normal coagulation proteome composition. [ABSTRACT FROM AUTHOR]

Factor (f)Xa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473) and control (n = 426) population from the Leiden Thrombophilia Study and each individual's plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC), the maximum rate (MaxR) and level (MaxL) and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use). The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3-6 fold) in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90th percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6). Grouping defined by oral contraceptive (OC) use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals. [ABSTRACT FROM AUTHOR]

The view that clot time-based assays do not provide a sufficient assessment of an individual's hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarintreated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants. [ABSTRACT FROM AUTHOR]

Abstract: Background: Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371–3382). Methods: In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. Results and conclusions: Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. Conclusion: These observations may attribute to the difference in the ability of TF–FVIIa complex to activate factor X (FX). General significance: Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF. [Copyright &y& Elsevier]

Abstract: Introduction: More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events. Materials and Methods: We studied (1) 100 patients with acute ischemic stroke (n=50) or transient ischemic attack (TIA) (n=50) within the first 24hours from symptom onset, and (2) 100 individuals 1 to 4years following ischemic stroke (n=50) or TIA (n=50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI). Results: The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p≤0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p<0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced. Conclusion: Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF). Objective: To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc). Methods: The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays. Results: For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22±0.56nM; S.D.) is substantially higher than that for FVIIIc (0.65±0.29nM) and the chromogenic assay (FVIIIch; 0.50±0.23nM). A positive correlation between FVIIIag and VWFag with R2 =0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77±0.31 vs. 1.14±0.48nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87±0.36 and 0.94±0.44nM, respectively. Conclusions: 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag. [Copyright &y& Elsevier]

The function of tissue factor (Tf)-initiated coagulation is hemorrhage control through the formation and maintenance of an impermeable platelet-fibrin barrier. The catalytic processes involved in the clot maintenance function are not well defined, although the rebleeding problems characteristic of individuals with hemophilias A and B suggest a link between specific defects in the Tf-initiated process and defects in the maintenance function. We have previously demonstrated, using a methodology of "flow replacement" (or resupply) of ongoing Tf-initiated reactions with fresh reactants, that procoagulant complexes are produced during Tf-initiated coagulation, which are capable of reinitiating coagulation without input from extrinsic factor Xase activity (Orfeo, T., Butenas, S., Brummel-Ziedins, K. E., and Mann, K. G. (2005)1. Biol. Chem. 280, 42887-42896). Here we used Tf-initiated reactions in normal and hemophilia blood or in their corresponding proteome mixtures as sources of pro-coagulant end products and then varied the resupplying mate- rial to determine the identity of the catalysts that drive the new cycle of thrombin formation. The central findings are as follows: 1) the prothrombinase complex (fVa-fXa-Ca2+-membrane) accumulated during the episode of Tf-initiated coagulation is the primary catalyst responsible for the observed pattern of prothrombin activation after resupply; 2) impairments in intrinsic factor Xase function, i.e. hemophilias A and B, result in an impaired capacity to mount a resupply response; and 3) in normal hemostasis the intrinsic factor Xase function contributes to the durability of the resupply response. [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. [Copyright &y& Elsevier]

Abstract: Tissue factor (TF) is the major physiological initiator of blood coagulation. It exists as an integral membrane protein that upon injury to the blood vessel becomes exposed to the blood stream and initiates a series of enzymatic reactions that cause blood to clot. TF is also found within circulating blood cells, requiring specific signaling events to promote its expression. In this review we will discuss current controversies concerning the structure–activity relationships of TF and contributions of TF to the hemostatic process, the potential roles of intravascular TF, including non-cell-bound TF and cell-expressed TF and the overall relationship between TF function and hemorrhage control. [Copyright &y& Elsevier]

Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (‘flow replacement’). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240 s); and a period in which the progress of the reaction is TF independent (>240s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists. [ABSTRACT FROM AUTHOR]

The presence of activation peptides (AP) of the vitamin K-dependent proteins in the phlebotomy blood of human subjects suggests that active serine proteases may circulate in blood as well. The goal of the current study was to evaluate the influence of trace amounts of key coagulation proteases on tissue factor-independent thrombin generation using three models of coagulation. With procoagulants and select coagulation inhibitors at mean physiological concentrations, concentrations of factor IXa, factor Xa, and thrombin were set either equal to those of their AP or to values that would result based upon the rates of AP/enzyme generation and steady state enzyme inhibition. In the latter case, numerical simulation predicts that sufficient thrombin to produce a solid clot would be generated in ∼2 min. Empirical data from the synthetic plasma suggest clotting times of 3-5 min, which are similar to that observed in contact pathway-inhibited whole blood (4.3 min) initiated with the same concentrations of factors IXa and Xa and thrombin. Numerical simulations performed with the concentrations of two of the enzymes held constant and one varied suggest that the presence of any pair of enzymes is sufficient to yield rapid clot formation. Modeling of states (numerical simulation and whole blood) where only one circulating protease is present at steady state concentration shows significant thrombin generation only for factor IX. The addition of factor Xa and thrombin has little effect (if any) on thrombin generation induced by factor IXa alone. These data indicate that 1) concentrations of active coagulation enzymes circulating in vivo are significantly lower than can be predicted from the concentrations of their AP, and 2) expected trace amounts of factor IXa can trigger thrombin generation in the absence of tissue factor. [ABSTRACT FROM AUTHOR]

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V actiration are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation, α-Thrombin, derived from factor Xa cleavage of phospholipidbound prothrombin via the prothrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation. [ABSTRACT FROM AUTHOR]

Objectives: To assess the effects of invasive procedures, hemostatic and clinical variables, the timing of beta-blocker therapy, and the doses of recombinant plasminogen activator (rt-PA) on hemorrhagic events.Design: A multicenter, randomized, controlled trial.Setting: Hospitals participating in the Thrombolysis in Myocardial Infarction, Phase II trial (TIMI II).Interventions: Patients received rt-PA, heparin, and aspirin. The total dose of rt-PA was 150 mg for the first 520 patients and 100 mg for the remaining 2819 patients. Patients were randomly assigned to an invasive strategy (coronary arteriography with percutaneous angioplasty [if feasible] done routinely 18 to 48 hours after the start of thrombolytic therapy) or to a conservative strategy (coronary arteriography done for recurrent spontaneous or exercise-induced ischemia). Eligible patients were also randomly assigned to either immediate intravenous or deferred beta-blocker therapy.Measurements: Patients were monitored for hemorrhagic events during hospitalization.Main Results: In patients on the 100-mg rt-PA regimen, major and minor hemorrhagic events were more common among those assigned to the invasive than among those assigned to the conservative strategy (18.5% versus 12.8%, P less than 0.001). Major or minor hemorrhagic events were associated with the extent of fibrinogen breakdown, peak rt-PA levels, thrombocytopenia, prolongation of the activated partial thromboplastin time (APTT) to more than 90 seconds, weight of 70 kg or less, female gender, and physical signs of cardiac decompensation. Immediate intravenous beta-blocker therapy had no important effect on hemorrhagic events when compared with delayed beta-blocker therapy. Intracranial hemorrhages were more frequent among patients treated with the 150-mg rt-PA dose than with the 100-mg rt-PA dose (2.1% versus 0.5%, P less than 0.001). The extent of the plasmin-mediated hemostatic defect was also greater in patients receiving the 150-mg dose.Conclusions: Increased morbidity due to hemorrhagic complications is associated with an invasive management strategy in patients with acute myocardial infarction. Our findings show the complex interaction of several factors in the occurrence of hemorrhagic events during thrombolytic therapy. [ABSTRACT FROM AUTHOR]

Presents information on a study that examined bone loss in patients with primary biliary cirrhosis. Methodology of the study; Results and discussion on the study.