Kathleen E. Brummel-Ziedins

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

Summary.  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 °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 ≤ 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.

Statins and Blood Coagulation

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. There is growing evidence that treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. Accordingly, statin use has been associated with impairment of several coagulant reactions catalyzed by this enzyme. Moreover, evidence indicates that statins, via increased thrombomodulin expression on endothelial cells, may enhance the activity of the protein C anticoagulant pathway. Most of the antithrombotic effects of statins are attributed to the inhibition of isoprenylation of signaling proteins. These novel properties of statins, suggesting that these drugs might act as mild anticoagulants, may explain, at least in part, the therapeutic benefits observed in a wide spectrum of patients with varying cholesterol levels, including subjects with acute coronary events.

Thrombin generation profiles in deep venous thrombosis.

Summary.  Background: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. Objective: Using an integrated approach that defines an individuals 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 ≤ 45 years>OR:2.5), BMI (OR:2.9 ≤ 26 kg m−2>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 individuals blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.

Thrombin generation and whole blood viscoelastic assays in the management of hemophilia: current state-of-art and future perspectives

Hemophilia is a bleeding disorder that afflicts about 1 in 5000 males. Treatment relies upon replacement of the deficient factor, and response to treatment both in clinical research and practice is based upon subjective parameters such as pain and joint mobility. Existing laboratory assays quantify the amount of factor in plasma, which is useful diagnostically and prognostically. However, these assays are limited in their ability to fully evaluate the patients clot-forming capability. Newer assays, known as global assays, provide a far more detailed view of thrombin generation and clot formation and have been studied in hemophilia for about 10 years. They have the potential to offer a more objective measure of both the hemophilic phenotype as well as the response to treatment. In particular, in patients who develop inhibitors to deficient clotting factors and in whom bypassing agents are required for hemostasis, these assays offer the opportunity to determine the laboratory response to these interventions where traditional coagulation assays cannot. In this article we review the existing literature and discuss several controversial issues surrounding the assays. Last, a vision of future clinical uses of these assays is briefly described.

The Tissue Factor Requirement in Blood Coagulation

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 (<10 s); 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 (>240 s). 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.

Thrombin generation: phenotypic quantitation

Summary.  An individuals 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 µg 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 individuals 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 individuals 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 individuals response to a tissue factor challenge.

Platelets in Tumor Progression: A Host Factor That Offers Multiple Potential Targets in the Treatment of Cancer

While platelets are well known to play a central role in hemostasis and thrombosis, there is emerging experimental evidence to suggest that they also mediate tumor cell growth, dissemination, and angiogenesis. An increase in platelet number (thrombocytosis) and activity is seen in patients with a wide spectrum of malignancies, and the former is correlated with a decrease in overall survival and poorer prognosis. Preclinical data suggest that circulating tumor cell partnerships with platelets in the blood facilitate tumor metastases through direct interactions and secreted bioactive proteins. Platelets form aggregates with tumor cells, thereby protecting them from host immune surveillance through physical shielding and induction of “platelet mimicry.” There is also laboratory evidence to suggest that activated platelets interact with cancer cells within the tumor microenvironment through paracrine signaling and direct contact, thereby promoting tumor cell growth and survival. For example, platelets release mediators of both tumor angiogenesis and osteoclast resorption. The interplay between platelets and tumor cells is complex and bidirectional with involvement of multiple other components within the tumor microenvironment, including immune cells, endothelial cells, and the extracellular matrix. We review the role of platelets in tumor progression, emphasizing the opportunity these interactions afford to target platelets and platelet function to improve patient outcomes in the cancer prevention and treatment setting. J. Cell. Physiol. 229: 1005–1015, 2014.

The plasma hemostatic proteome: thrombin generation in healthy individuals

Summary.  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 ∼ 4‐fold range (200–800 nm), maximum rates varied ∼ 4.8‐fold (90–435 nm min−1) and total thrombin varied ∼ 4.5‐fold (39–177 µm 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 individuals propensity for developing thrombosis.

Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition

Summary.  Background: Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). Methods and results: Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age‐ and sex‐matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual’s coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. Conclusion: Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD.

Systemic blood coagulation activation in acute coronary syndromes

We evaluated systemic alterations to the blood coagulation system that occur during a coronary thrombotic event. Peripheral blood coagulation in patients with acute coronary thrombosis was compared with that in people with stable coronary artery disease (CAD). Blood coagulation and platelet activation at the microvascular injury site were assessed using immunochemistry in 28 non-anticoagulated patients with acute myocardial infarction (AMI) versus 28 stable CAD patients matched for age, sex, risk factors, and medications. AMI was associated with increased maximum rates of thrombin-antithrombin complex generation (by 93.8%; P< .001), thrombin B-chain formation (by 57.1%; P< .001), prothrombin consumption (by 27.9%; P= .012), fibrinogen consumption (by 27.0%; P= .02), factor (f) Va light chain generation (by 44.2%; P= .003), and accelerated fVa inactivation (by 76.1%; P< .001), and with enhanced release of platelet-derived soluble CD40 ligand (by 44.4%; P< .001). FVa heavy chain availability was similar in both groups because of enhanced formation and activated protein C (APC)-mediated destruction. The velocity of coagulant reactions in AMI patients showed positive correlations with interleukin-6. Heparin treatment led to dampening of coagulant reactions with profiles similar to those for stable CAD. AMI-induced systemic activation of blood coagulation markedly modifies the pattern of coagulant reactions at the site of injury in peripheral vessels compared with that in stable CAD patients.