Rinske Loeffen

The impact of blood coagulability on atherosclerosis and cardiovascular disease

Summary.  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.

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

Summary.  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.

Associations Between Thrombin Generation and the Risk of Cardiovascular Disease in Elderly Patients: Results From the PROSPER Study

BACKGROUND Hypercoagulability may be an important contributor to the pathophysiology of atherosclerosis and atherothrombosis. As thrombin fulfills a central role in coagulation and links to several cellular mechanisms involved in arterial disease, we hypothesized that thrombin generation is associated with cardiovascular events in elderly patients. METHODS We studied the relationship between plasma thrombin generation and incident coronary heart disease (CHD) and stroke in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). From this multicenter prospective cohort, 4,932 samples of subjects (70-82 years) with pre-existing vascular disease or risk factors were available for thrombin generation measurements. RESULTS Within the 3.2 years of follow-up incident stroke and CHD was observed in 227 and 545 subjects, respectively. Baseline thrombin generation was significantly decreased in subjects with incident stroke compared with subjects without: normalized peak height 71.1±40.8% versus 82.3±44.9%, p = .0002, and normalized endogenous thrombin potential 79.1±23.3% versus 87.0±24.8%, p < .0001 (mean and SDs). Thrombin generation was independently and inversely associated with stroke risk: hazard ratio 0.71 (95%CI: 0.60-0.85), 0.68 (95%CI: 0.58-0.79), for normalized peak height and normalized endogenous thrombin potential, respectively (all p < .001). In subjects with incident CHD, thrombin generation was comparable to subjects without a coronary event. Only an increased normalized peak height was significantly associated with incident CHD (hazard ratio 1.17 [95% CI: 1.06-1.28], p = .002). CONCLUSIONS We demonstrate that a delayed and decreased thrombin generation is a strong and independent predictor for stroke in elderly people at increased risk of vascular disease. However, no convincing consistent association could be demonstrated between thrombin generation and incident CHD.

Long-term strenuous exercise induces a hypercoagulable state through contact activation

Jelle J. Posthuma1; Rinske Loeffen1; Rene van Oerle1,2; Yvonne M. C. Henskens2; Hugo ten Cate1; Henri M. H. Spronk1; Paola E. J. van der Meijden1 1Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, Netherlands; 2Haemostasis Laboratory, Department of Internal Medicine, Maastricht University Medical Center, Maastricht, Netherlands

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.

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.

Intermediate and Nonclassical Monocytes Show Heterogeneity in Patients with Different Types of Acute Coronary Syndrome

This study was performed to gain further insight in the heterogeneity of monocytes in the different categories of acute coronary syndrome (ACS), especially between patients with unstable angina pectoris, ST‐elevation myocardial infarction (STEMI), and non‐ST‐elevation myocardial infarction (NSTEMI). For this purpose, blood samples were collected in the acute phase from patients presenting with an ACS. These samples were examined with multiparameter flow cytometry to identify the different monocyte subsets and to analyze the expression of monocyte‐associated molecules. Leukocytes, as well as an absolute number of monocytes, showed a clear and significant increase in patients with STEMI. This increase was seen in all subtypes of monocytes. The classical monocytes (CD14++CD16–) of patients with an NSTEMI had a significantly increased CD11b expression when compared to the control group, while these cells showed a decreased expression pattern in STEMI patients. This increased CD11b‐expression was also seen in the intermediate monocytes of NSTEMI, while it was almost completely downregulated on the intermediate monocytes of STEMI. Finally, CX3CR1, which is almost exclusively expressed on intermediate and nonclassical monocytes, showed a significant decrease in expression in patients with STEMI. In conclusion, intermediate and nonclassical monocytes have a different immunophenotypic pattern in patients with STEMI versus NSTEMI. These differences reflect the pro‐inflammatory state of the monocytes in NSTEMI and can be used as target molecules for novel therapeutic strategies to diminish the migration of proinflammatory monocytes into the myocardial tissue.

Factor XIa and Thrombin Generation Are Elevated in Patients with Acute Coronary Syndrome and Predict Recurrent Cardiovascular Events.

Objective In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS. Methods We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis. Results Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevated in ACS patients: 1.9±1.1 vs. 1.4±0.7 pM and 495(310–885) vs. 380(235–540) μg/L. Within the ACS spectrum, ST-elevated myocardial infarction patients had the highest prothrombotic profile. During the acute event, thrombin generation was significantly increased compared to 1 and 6 months afterwards: peak height 145±52 vs. 100±44 vs. 98±33 nM. Both peak height and factor XIa levels on admission predicted recurrent cardiovascular events (OR: 4.9 [95%CI 1.2–20.9] and 4.5 [1.1–18.9]). Conclusion ACS patients had an enhanced prothrombotic profile, demonstrated by an increased thrombin generation potential, factor XIa and D-dimer levels. This study is the first to demonstrate the positive association between factor XIa, thrombin generation and recurrent cardiovascular events.

The impact of blood coagulability on atherosclerosis and cardiovascular disease: reply to a rebuttal: Letters to the Editor

In their letter Clotting factors vs. lipids in the pathogenesis of atherosclerosis: a lost cause for the former Girolami et al. address several issues raised by our recent review article The impact of blood coagulability on atherosclerosis and cardiovascular disease [1]. First of all, the authors claim that congenital (chronic) hypocoagulability does not protect against atherosclerosis, as demonstrated by several clinical and laboratory observations. As clearly pointed out in our review, the traditional view of congenital hypocoagulability being protective against cardiovascular disease is indeed considered to be controversial, with several studies providing evidence for [2–5] and against [6–10] the hypothesis. It should, however, be noted that, in contrast to the experimental work, hemophilic patients cannot be considered to be null for the respective clotting factors, as either they are transfused (and survive nowadays to the stage when they do acquire atherosclerosis), or their disease is mild to moderate, which raises questions about the degree of protection that such deficiencies may offer against a complex process such as atherosclerosis. Second, although the presence of coagulation factors within atherosclerotic lesions does not imply a causal relationship between the two, there is strong evidence for involvement of the coagulation system in the development and progression of atherosclerotic disease. Wilcox et al. showed the direct synthesis (mRNA expression) of coagulation proteins in the arterial vessel wall by both smooth muscle cells and macrophages [11,12]. As indicated in our review, a second source for coagulation factors may indeed be uptake from the circulation. Besides the presence of coagulation proteins, fibrinogen and fibrin degradation products in atherosclerotic lesions [13–15], it was demonstrated that plaque-derived clotting proteins were functionally active in an in vitro thrombin generation assay [16], and they were shown to be associated with the stage and severity of atherosclerotic disease [16]. Furthermore, many complex processes involved in the development and progression of atherosclerosis, including angiogenesis, inflammation, and cell proliferation, are regulated by the activation of protease-activated receptors (PARs) on vascular cells. Therefore, by activating PARs, active coagulation proteins may have profound implications for atherogenesis [17]. On the basis of these results, we derive the hypothesis that the coagulation system is a potential contributor to the pathogenesis of atherosclerosis. Third, Girolami et al. state that anticoagulant therapies are unable to modify the structure and progression of atherosclerotic plaques. This assertion is, however, contradicted by recent animal studies [18–20] that investigated whether modulation of thrombin activity by direct thrombin or factor Xa inhibitors would affect atherosclerotic progression. Kadoglou et al. provided evidence that long-term treatment with dabigatran in addition to a high-fat diet reduced atherosclerotic progression in apolipoprotein E (ApoE) mice, despite the absence of blood lipid changes [20]. Furthermore, dabigatran seemed to promote plaque stability, probably bymodifying inflammatory mechanisms. In our own studies, we observed similar inhibitory effects of dabigatran on atherosclerosis in mice with the severe thrombomodulin (TM)/ApoE phenotype, also related to anti-inflammatory activity [21]. Moreover, these two studies confirm the results of previous work performed with the thrombin inhibitor melagatran [18]. Although these studies show convincing evidence for anticoagulant-induced atherosclerosis reduction in animals, the effects in patients with preexisting atherosclerosis have still to be determined. Fourth, we agree with Girolami et al. in that a clear distinction should be made between atherosclerosis and atherothrombosis. Therefore, throughout the whole review, we used the term atherosclerosis for the development and progression of streaks and plaques within the arterial vessel wall, and arterial thrombosis for pathologic thrombus formation. Finally, to conclude, there is involvement of more than lipids with regard to the pathogenesis of atherosclerosis, with abundant experimental evidence suggesting clear biological effects of coagulation factors on the progression of atheroscleCorrespondence: Rinske Loeffen, Laboratory for Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, P.O. Box 616, UNS50: Box8, 6200 MD Maastricht, the Netherlands. Tel.: +31 43388 4262; fax: +31 43388 4159. E-mail: rinske.loeffen@maastrichtuniversity.nl