The hypercoagulable profile of patients with stent thrombosis.

OBJECTIVE: Coronary stent thrombosis is a devastating complication after percutaneous coronary intervention (PCI). The mechanisms underlying stent thrombosis are multifactorial. Whether the coagulation system is involved in the pathophysiology of stent thrombosis is unclear. We hypothesised that thrombin generation, reflecting the coagulation potential, is enhanced in patients with stent thrombosis. METHODS: A case-control study was performed, including 63 patients with PCI: 23 cases (stent thrombosis) and 40 controls (no stent thrombosis). Thrombin generation was measured using 0, 1 and 5 pM tissue factor (TF) triggers. Active site-inhibited factor VIIa (ASIS) and recombinant thrombomodulin were added to study the contact activation system and the protein C pathway, respectively. RESULTS: Thrombin generation was significantly increased for all TF triggers in cases compared with controls. Addition of ASIS to the measurement without exogenous TF revealed significantly enhanced contact activation in cases compared with controls; mean peak height: 241 vs 183 nM. Thrombin generation was also significantly increased in cases compared with controls in the presence of exogenous TF; mean peak height: 263 vs 233 nM (5 pM TF). Addition of thrombomodulin reduced thrombin generation by 23% in cases and 31% in controls (p<0.018), suggesting alterations in the protein C pathway in cases. CONCLUSIONS: This is the first study that suggests the involvement of the coagulation system in stent thrombosis. Stent thrombosis patients showed a hypercoagulable state, most likely caused by enhanced contact activation and attenuation of anticoagulation by the protein C pathway.

Increased factor XIa levels in patients with a first acute myocardial infarction: the introduction of a new thrombin generation based factor XIa assay.

OBJECTIVE: One of the contributing mechanisms in acute myocardial infarction (AMI) is plasma hypercoagulability. Recently, it was suggested that factor XI activation might play a role in atherothrombosis. To quantify factor XIa plasma levels, we developed a new thrombin generation based assay and hypothesized that in AMI patients factor XIa levels are increased during the acute thrombotic event. METHODS: A prospective cohort study was performed including 56 patients with first AMI. Blood was collected upon admission and after 6 months. Reference blood samples were obtained from 30 apparently healthy control subjects. Plasma samples were diluted (1:5) in factor XI deficient plasma and factor XIa plasma levels were established using a reference curve (0-12.5 pM factor XIa) and an inhibitory anti-factor XIa antibody. The established FXIa concentrations were related to the 1-year outcome. RESULTS: Factor XIa plasma concentrations were significantly increased in AMI patients on admission compared to 6 months after the event (3.7 pM [2.7-5.5] vs. 2.8 [1.9-4.3], median ± IQR; P=0.001) and compared to healthy controls (3.7 pM [2.7-5.5] vs. 2.7 [1.6-4.2], median ± IQR; P=0.004). However, a high factor FXIa level at baseline was not significantly associated with a recurrent cardiovascular event (OR 1.26, 95%CI 0.33-4.7). CONCLUSIONS: This study presents the first application of a new thrombin generation based factor XIa assay, showing significantly increased factor XIa levels in AMI patients on admission compared to 6 months after the event and compared to healthy controls. The factor XIa concentration was not associated with the risk of recurrence.

Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method.

BACKGROUND: Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method. METHODS AND RESULTS: Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10,000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of < 15% for all derived parameters with both the 1 and 5 pm TF triggers. CONCLUSION: Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.

The impact of blood coagulability on atherosclerosis and cardiovascular disease.

Although the link between blood coagulation and atherogenesis has been long postulated, only recently, and through the extensive work on transgenic mice, crossbred on an atherogenic background, has the direction of this interaction become visible. In general, hypercoagulability in mice tends to increase atherosclerosis, whereas hypocoagulability reduces the atherosclerotic burden, depending on the mouse model used. The information on a direct relationship between coagulation and atherosclerosis in humans, however, is not that clear. Almost all coagulation proteins, including tissue factor, are found in atherosclerotic lesions in humans. In addition to producing local fibrin, a matrix for cell growth, serine proteases such as thrombin may be very important in cell signaling processes, acting through the activation of protease-activated receptors (PARs). Activation of PARs on vascular cells drives many complex processes involved in the development and progression of atherosclerosis, including inflammation, angiogenesis, and cell proliferation. Although current imaging techniques do not allow for a detailed analysis of atherosclerotic lesion phenotype, hypercoagulability, defined either by gene defects of coagulation proteins or elevated levels of circulating markers of activated coagulation, has been linked to atherosclerosis-related ischemic arterial disease. New, high-resolution imaging techniques and sensitive markers of activated coagulation are needed in order to study a causal contribution of hypercoagulability to the pathophysiology of atherosclerosis. Novel selective inhibitors of coagulation enzymes potentially have vascular effects, including inhibition of atherogenesis through attenuation of inflammatory pathways. Therefore, we propose that studying the long-term vascular side effects of this novel class of oral anticoagulants should become a clinical research priority.

Thigh-calf contact force measurements in deep knee flexion.

BACKGROUND: Knee models often do not contain thigh-calf contact which occurs in deep knee flexion. Thigh-calf contact is expected to reduce muscle forces and thereby affects internal stresses in the knee joint. The purpose of this study was to measure thigh-calf contact forces. Two deep knee flexion activities were selected: squatting and kneeling. METHODS: Ten healthy subjects participated in the experiment. Contact pressures between the thigh and calf were measured using the Tekscan Conformat pressure mapping sensor. Knee flexion angles were measured unilaterally using an infrared motion capture system. Contact forces were averaged in terms of means and standard deviations. The magnitude and location of the resultant contact force were calculated. Correlations between anthropometric subject parameters and experimental outcome were studied. FINDINGS: In general, thigh-calf contact did not take place below 130 degrees knee flexion. The average maximal contact forces for each leg were 34.2% bodyweight during squatting and 30.9% bodyweight during kneeling. Corresponding average maximal knee flexion angles were 151.8 degrees during squatting and 156.4 degrees during kneeling. Thigh and calf circumferences were correlated with the contact force measurements. INTERPRETATION: The current study shows that thigh-calf contact is substantial (>30% bodyweight on one leg) and likely reduces the forces inside the knee during deep knee flexion. Subsequently, total knee replacements may be subjected to lower loads than assumed before, which reduces the risk of implant failure at large flexion angles. Results presented in this study can be utilized in knee models that focus on deep knee flexion.

Synergism between androgens and estrogens in the induction of aromatase and its messenger RNA in the brain.

It is established that testosterone (T) increases aromatase activity (AA) in the quail brain and that this induction of AA represents a limiting factor in the activation of male copulatory behavior. This action of T presumably results from an induction of aromatase synthesis since the number of aromatase-immunoreactive (ARO-ir) cells increases and, in parallel, there is an increase in aromatase mRNA as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) technology. The specific role of androgenic and estrogenic metabolites of T in this induction is not yet clear but product-formation assays suggest that both types of compounds synergize to increase AA. The exact role of androgens and estrogens in the induction of aromatase was examined by studying both the aromatase protein by immunocytochemistry and the aromatase mRNA by RT-PCR in castrated quail that had been treated with T or its androgenic metabolite, 5 alpha-dihydrotestosterone (DHT) or its estrogenic metabolite, estradiol-17 beta (E2) or both DHT and E2 simultaneously. A specific quantitative PCR technique using a modified aromatase as internal standard was developed for this purpose. T increased the number of ARO-ir cells in all brain areas and increased the concentration of ARO mRNA in the preoptic area-anterior hypothalamus (POA-aHYP) and in the posterior hypothalamus (pHYP). E2-treated birds had more ARO-ir cells than castrates in the posterior part of the medial preoptic nucleus (POM), in the bed nucleus stria terminalis (BNST) and tuber. Their aromatase mRNA concentration was significantly increased in the POA-aHYP but this effect did not reach significance in the pHYP. DHT by itself had no effect on either the number of ARO-ir cells (all brain regions considered) or the concentration of aromatase mRNA. DHT, however, synergized with E2, both in inducing ARO-ir neurons and in increasing aromatase mRNA concentration. This synergism was shown to be statistically significant in several brain areas. These data demonstrate that both androgens and estrogens regulate aromatase at the pretranslational level. Because the percentage increase in the number of ARO-iR cells was in general very similar to the increase in aromatase mRNA concentration, these data also suggest that these steroids regulate aromatase mostly by changing its mRNA synthesis or catabolism.