Plasma protein signatures for high on-treatment platelet reactivity to aspirin and clopidogrel in peripheral artery disease.

BACKGROUND: A significant proportion of patients with peripheral artery disease (PAD) displays a poor response to aspirin and/or the platelet P2Y12 receptor antagonist clopidogrel. This phenomenon is reflected by high on-treatment platelet reactivity (HTPR) in platelet function assays in vitro and is associated with an increased risk of adverse cardiovascular events. OBJECTIVE: This study aimed to elucidate specific plasma protein signatures associated with HTPR to aspirin and clopidogrel in PAD patients. METHODS AND RESULTS: Based on targeted plasma proteomics, 184 proteins from two cardiovascular Olink panels were measured in 105 PAD patients. VerifyNow ASPI- and P2Y12-test values were transformed to a continuous variable representing HTPR as a spectrum instead of cut-off level-defined HTPR. Using the Boruta random forest algorithm, the importance of 3 plasma proteins for HTPR in the aspirin, six in clopidogrel and 10 in the pooled group (clopidogrel or aspirin) was confirmed. Network analysis demonstrated clusters with CD84, SLAMF7, IL1RN and THBD for clopidogrel and with F2R, SELPLG, HAVCR1, THBD, PECAM1, TNFRSF10B, MERTK and ADM for the pooled group. F2R, TNFRSF10B and ADM were higher expressed in Fontaine III patients compared to Fontaine II, suggesting their relation with PAD severity. CONCLUSIONS: A plasma protein signature, including eight targets involved in proatherogenic dysfunction of blood cell-vasculature interaction, coagulation and cell death, is associated with HTPR (aspirin and/or clopidogrel) in PAD. This may serve as important systems-based determinants of poor platelet responsiveness to aspirin and/or clopidogrel in PAD and other cardiovascular diseases and may contribute to identify novel treatment strategies.

Diabetes- versus smoking-related thrombo-inflammation in peripheral artery disease.

Peripheral artery disease (PAD) is a major health problem with increased cardiovascular mortality, morbidity and disabling critical limb threatening ischemia (CLTI) and amputation. Diabetes mellitus (DM) and cigarette smoke are the main risk factors for the development of PAD. Although diabetes related PAD shows an accelerated course with worse outcome regarding complications, mortality and amputations compared with non-diabetic patients, current medical treatment does not make this distinction and includes standard antiplatelet and lipid lowering drugs for all patients with PAD. In this review we discuss the pathophysiologic mechanisms of PAD, with focus on differences in thrombo-inflammatory processes between diabetes-related and smoking-related PAD, and hypothesize on possible mechanisms for the progressive course of PAD in DM. Furthermore, we comment on current medical treatment and speculate on alternative medical drug options for patients with PAD and DM.

Inadequate response to antiplatelet therapy in patients with peripheral artery disease: a prospective cohort study.

BACKGROUND: Patients with peripheral artery disease (PAD) are treated with preventive strategies to improve the cardiovascular risk. The incidence of cardiovascular events and mortality however remains high in PAD populations. We therefore aimed to better characterize PAD patients suffering from cardiovascular events and mortality in order to tailor preventive treatment. METHODS: Between 2018 and 2020, 246 PAD outpatients (17 newly diagnosed, 229 with known PAD) were prospectively enrolled in this observational cohort study. Patient data and blood samples were collected after inclusion, and the primary composite endpoint (myocardial infarction, elective coronary revascularization, ischemic stroke, acute limb ischemia, mortality) was evaluated after one year. Secondary outcomes included platelet reactivity, measured using the VerifyNow assay, and medication adherence, assessed using the Morisky Medication Adherence Scale-8 (MMAS-8). Logistic regression models were used to identify associations between characteristics and the occurrence of events. RESULTS: The cohort comprised 207 patients with claudication and 39 with chronic limb threatening ischemia. Twenty-six (10.6%) patients suffered from an event during follow-up. Prior myocardial infarction (OR 3.3 [1.4-7.7]), prior ischemic stroke (OR 4.5 [1.8-10.9]), higher levels of creatinine (OR 5.2 [2.2-12.6]), lower levels of high-density lipoprotein (OR 4.2 [1.5-10.6]) and lower haemoglobin levels (OR 3.1 [1.3-7.1]) were associated with events. Patients with events had more often high on-treatment platelet reactivity (HTPR) on aspirin (OR 5.9 [1.4-25.1]) or clopidogrel (OR 4.3 [1-19.3]). High adherence to medication was associated with the occurrence of events (OR 4.1 [1-18]). CONCLUSIONS: Patients suffering from cardiovascular events and mortality were characterized by prior cardiovascular events as compared to patients who did not experience any events. Antiplatelet therapy was not optimally protective despite high medication adherence, and HTPR was independently associated with the occurrence of events. More research is needed on alternative treatment strategies such as dual antiplatelet therapy or combinations with anticoagulant drugs. TRIAL REGISTRATION: The Medical Ethics Committee (METC) of the MUMC+ approved the study (NL63235.068.17) and the study was registered in the Netherlands Trial Register ( NTR7250 ).

Translational insights into mechanisms underlying residual venous obstruction and the role of factor XI, P-selectin and GPVI in recurrent venous thromboembolism.

BACKGROUND: Residual venous obstruction (RVO) after deep vein thrombosis (DVT) is considered a risk factor of recurrent venous thromboembolism (VTE), arterial events and post-thrombotic syndrome (PTS). We hypothesized thrombo-inflammatory markers might be associated with RVO and clinical outcomes. MATERIALS AND METHODS: In a DVT cohort with routine RVO-assessment and 5-year follow-up, patients were invited for blood withdrawal after stopping anticoagulants. Thrombin generation potential, coagulation enzyme:inhibitor complexes, soluble platelet markers and clinical markers were measured in platelet-poor plasma. Associations were represented as odds ratio (OR) or hazard ratio (HR) per standard deviation. RESULTS: Patients with RVO (102/306, 33 %) had higher rates of PTS (24 vs. 12 %, p = 0.008), but similar rates of recurrence (16 vs. 15 %, p = 0.91) and arterial events (7 vs. 4 %, p = 0.26). RVO was associated with thrombin peak height (OR 1.40 [1.04-1.88]), endogenous thrombin potential (ETP, OR 1.35 [1.02-1.79]), and CRP (OR 1.74 [1.10-2.75]). Recurrent VTE was associated with ETP (HR 1.36 [1.03-1.81]), FXIa:C1-inhibitor (HR 1.34 [1.04-1.72]), thrombin:antithrombin (HR 1.36 [1.16-1.59]), soluble P-selectin (HR 2.30 [1.69-3.11]), soluble glycoprotein VI (sGPVI, HR 1.30 [1.01-1.69]), D-dimer (HR 1.56 [1.31-1.86]), and factor VIII (HR 1.44 [1.15-1.82]). Arterial events were associated with sGPVI (HR 1.80 [1.25-2.59]). PTS was not associated with any marker. CONCLUSIONS: Our findings indicate RVO was associated with thrombo-inflammation, but this did not predict clinical outcomes in this setting. Importantly, we found recurrent VTE was associated with ongoing coagulation and platelet activation in patients well beyond the acute phase of DVT. Furthermore, sGPVI indicated an increased risk of arterial events, highlighting the role of platelets in arterial thrombosis following DVT.

Discovery of four plasmatic biomarkers potentially predicting cardiovascular outcome in peripheral artery disease.

Peripheral artery disease (PAD) patients have an increased cardiovascular risk despite pharmacological treatment strategies. Biomarker research improving risk stratification only focused on known atherothrombotic pathways, but unexplored pathways might play more important roles. To explore the association between a broad cardiovascular biomarker set and cardiovascular risk in PAD. 120 PAD outpatients were enrolled in this observational cohort study. Patients were followed for one year in which the composite endpoint (myocardial infarction, coronary revascularization, stroke, acute limb ischemia and mortality) was assessed. Patient data and blood samples were collected upon inclusion, and citrated platelet-poor plasma was used to analyze 184 biomarkers in Olink Cardiovascular panel II and III using a proximity extension assay. Fifteen patients reached the composite endpoint. These patients had more prior strokes and higher serum creatinine levels. Multivariate analysis revealed increased plasma levels of protease-activated receptor 1 (PAR1), galectin-9 (Gal-9), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) and interleukin 6 (IL-6) to be most predictive for cardiovascular events and mortality. Positive regulation of acute inflammatory responses and leukocyte chemotaxis were identified as involved biological processes. This study identified IL-6, PAR1, Gal-9, TNFRSF11A as potent predictors for cardiovascular events and mortality in PAD, and potential drug development targets.

Antithrombotic Therapy: Prevention and Treatment of Atherosclerosis and Atherothrombosis.

Atherosclerosis is a multifactorial vascular disease that develops in the course of a lifetime. Numerous risk factors for atherosclerosis have been identified, mostly inflicting pro-inflammatory effects. Vessel injury, such as occurring during erosion or rupture of atherosclerotic lesions triggers blood coagulation, in attempt to maintain hemostasis (protect against bleeding). However, thrombo-inflammatory mechanisms may drive blood coagulation such that thrombosis develops, the key process underlying myocardial infarction and ischemic stroke (not due to embolization from the heart). In the blood coagulation system, platelets and coagulation proteins are both essential elements. Hyperreactivity of blood coagulation aggravates atherosclerosis in preclinical models. Pharmacologic inhibition of blood coagulation, either with platelet inhibitors, or better documented with anticoagulants, or both, limits the risk of thrombosis and may potentially reverse atherosclerosis burden, although the latter evidence is still based on animal experimentation.Patients at risk of atherothrombotic complications should receive a single antiplatelet agent (acetylsalicylic acid, ASA, or clopidogrel); those who survived an atherothrombotic event will be prescribed temporary dual antiplatelet therapy (ASA plus a P2Y12 inhibitor) in case of myocardial infarction (6-12 months), or stroke (<6 weeks), followed by a single antiplatelet agent indefinitely. High risk for thrombosis patients (such as those with peripheral artery disease) benefit from a combination of an anticoagulant and ASA. The price of gained efficacy is always increased risk of (major) bleeding; while tailoring therapy to individual needs may limit the risks to some extent, new generations of agents that target less critical elements of hemostasis and coagulation mechanisms are needed to maintain efficacy while reducing bleeding risks.

Sustained inflammation, coagulation activation and elevated endothelin-1 levels without macrovascular dysfunction at 3 months after COVID-19.

INTRODUCTION: Endothelial damage and thrombosis caused by COVID-19 may imperil cardiovascular health. More than a year since the WHO declared COVID-19 pandemic, information on its effects beyond the acute phase is lacking. We investigate endothelial dysfunction, coagulation and inflammation, 3 months post-COVID-19. MATERIALS AND METHODS: A cohort study was conducted including 203 patients with prior COVID-19. Macrovascular dysfunction was assessed by measuring the carotid artery diameter in response to hand immersion in ice-water. A historic cohort of 312 subjects served as controls. Propensity score matching corrected for baseline differences. Plasma concentrations of endothelin-1 were measured in patients post-COVID-19, during the acute phase, and in matched controls. Coagulation enzyme:inhibitor complexes and inflammatory cytokines were studied. RESULTS AND CONCLUSIONS: The prevalence of macrovascular dysfunction did not differ between the COVID-19 (18.6%) and the historic cohort (22.5%, RD -4%, 95%CI: -15-7, p = 0.49). Endothelin-1 levels were significantly higher in acute COVID-19 (1.67 ± 0.64 pg/mL) as compared to controls (1.24 ± 0.37, p < 0.001), and further elevated 3 months post-COVID-19 (2.74 ± 1.81, p < 0.001). Thrombin:antithrombin(AT) was high in 48.3%. Markers of contact activation were increased in 16-30%. FVIIa:AT (35%) and Von Willebrand Factor:antigen (80.8%) were elevated. Inflammatory cytokine levels were high in a majority: interleukin(IL)-18 (73.9%), IL-6 (47.7%), and IL-1ra (48.9%). At 3 months after acute COVID-19 there was no indication of macrovascular dysfunction; there was evidence, however, of sustained endothelial cell involvement, coagulation activity and inflammation. Our data highlight the importance of further studies on SARS-CoV-2 related vascular inflammation and thrombosis, as well as longer follow-up in recovered patients.

Calibrated automated thrombogram II: removing barriers for thrombin generation measurements.

BACKGROUND: Thrombin generation (TG) assessed by Calibrated Automated Thrombogram (CAT-I) reflects the overall capacity of plasma to generate thrombin, thus evaluating the balance between the anti- and procoagulant processes. However, with this method the calibrator curve is usually not measured until completion which has a severe impact on the calculation of the TG parameters, especially under conditions where almost all substrate is consumed. In addition, direct thrombin inhibitor (DTI) cannot be present in the calibration sample due to inhibition of the calibrator. We have developed a modified TG assay (CAT-II) and performed head-to-head comparison with the CAT-I method using the same fluorometer. Furthermore, we have compared our CAT-II method to a new automated TG instrument (ST®-Genesia) using the same calibration method. METHODS: TG was assessed with CAT-I and CAT-II using the same fulorometer and with ST®-Genesia in control plasma and plasma containing different anticoagulants (dabigatran, rivaroxaban, apixaban) and plasmas to which common interfering substances, bilirubin, hemoglobin and lipids were added. In CAT-I, calibration was against the same plasma containing calibrator in the presence of fluorogenic substrate (Z-GGR-AMC). In contrast, CAT-II method and ST®-Genesia used a standard concentration of thrombin in buffer and 7-amino-4-methylcoumarin (AMC) in a separate plasma sample for calibration. RESULTS: TG obtained from CAT-I using anticoagulant-free plasmas was lower compared with TG from CAT-II but both methods demonstrated an intra-assay variation less than 5% on all measured parameters. When comparing the two different calibration methods in the presence of different anticoagulants, a high correlation was seen in the presence of rivaroxaban and apixaban (R2 > 0.97), but not with dabigatran, a direct thrombin inhibitor. CAT-II method showed dose-dependent inhibition of TG in the presence of dabigatran, while CAT-I was not able to detect it. Both methods were able to correct for the interfering substances. CONCLUSIONS: Our results showed high similarity between the results of CAT-I and CAT-II method when it is applied in control plasmas and plasmas not inhibited with a direct thrombin inhibitor. Furthermore, both the CAT-II method and ST-Genesia using the same calibration method were able to detect the effect of all oral anticoagulants. Taken together, applying a new calibration method is a significant improvement for monitoring patients on direct thrombin inhibitors while not introducing any bias to results obtained on other types of samples.

Design and synthesis of a multivalent catch-and-release assay to measure circulating FXIa.

BACKGROUND: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification. METHODS: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems. RESULTS: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma. CONCLUSION: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing.

Tissue factor (:Factor VIIa) in the heart and vasculature: More than an envelope.

Blood coagulation comprises a complex cellular and molecular mechanism that maintains vascular integrity, protects against bleeding (hemostasis) and responds to injury. However, several elements of the coagulation system, including several coagulation factors and platelets, are also involved in other physiological and pathological processes. Tissue factor (TF) is a cell surface glycoprotein expressed in a vast variety of cell types and essential for hemostasis. Upon exposure of the TF-rich subendothelium to the blood stream, Factor VII (FVII) can bind to TF. TF subsequently facilitates the activation of FVII into activated FVII (FVIIa) thereby initiating the extrinsic coagulation pathway followed by the activation of FX and thrombin formation. Besides its hemostatic role in the vasculature, the TF:FVIIa pathway is active in many other compartments and organs where it can take part and mediate different physiological and pathological processes. The so-called non-hemostatic functions of TF:VIIa play a role in diverse processes such as inflammation, atherosclerosis and vascular and cardiac remodeling. This narrative review aims to reassess the most important and recent findings regarding the complex signaling pathways initiated by the TF:FVIIa complex, with an emphasis on the heart and blood vessels. Understanding how the mechanisms of TF:FVIIa signaling contribute to both physiological and pathological processes, is one of the keys to the development of new treatment strategies in cardiovascular disease.

Pleiotropic effects of the hemostatic system.

Atherothrombosis is characterized by the inflammatory process of atherosclerosis combined with a hypercoagulable state leading to superimposed thrombus formation. In atherosclerotic plaques, cell signaling can occur via protease-activated receptors (PARs), four of which have been identified so far (PAR1-PAR4). Proteases that are able to activate PARs can be produced systemically, but also at the sites of lesions, and they include thrombin and activated factor X. After PAR activation, downstream signaling can lead to both proinflammatory effects and a hypercoagulable state. Which specific effect occurs depends on the type of protease and activated PAR, and the site of activation. Hypercoagulable effects are mainly exerted through PAR1 and PAR4, whereas proinflammatory responses are mostly seen after PAR1 and PAR2 activation. PAR signaling pathways contribute to atherothrombosis, suggesting that inhibition of these pathways possibly prevents cardiovascular events based on this pathophysiological mechanism. In this review, we highlight the pathways by which PAR activation leads to proinflammatory responses and a hypercoagulable state. Furthermore, we give an overview of potential pharmacological treatment targets that promote vascular protection.

Atherothrombosis and Thromboembolism: Position Paper from the Second Maastricht Consensus Conference on Thrombosis.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

Coagulation and non-coagulation effects of thrombin.

Thrombin is a multifunctional serine protease produced from prothrombin, and is a key regulator in hemostatic and non-hemostatic processes. It is the main effector protease in primary hemostasis by activating platelets, and plays a key role in secondary hemostasis. Besides its well-known functions in hemostasis, thrombin also plays a role in various non-hemostatic biological and pathophysiologic processes, predominantly mediated through activation of protease-activated receptors (PARs). Depending on several factors, such as the concentration of thrombin, the duration of activation, the location of PARs, the presence of coreceptors, and the formation of PAR heterodimers, activation of the receptor by thrombin can induce different cellular responses. Moreover, thrombin can have opposing effects in the same cell; it can induce both inflammatory and anti-inflammatory signals. Owing to the complexity of thrombin's signal transduction pathways, the exact mechanism behind the dichotomy of thrombin is yet still unknown. In this review, we highlight the hemostatic and non-hemostatic functions of thrombin, and specifically focus on the non-hemostatic dual role of thrombin under various conditions and in relation to cardiovascular disease.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

BACKGROUND AND OBJECTIVES: In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied. MATERIALS AND METHODS: 10 Omniplasma and 10 quarantine plasma units were used to study different procoagulation, anticoagulation and fibrinolytic parameters. Analysis took place at different time-points during plasma storage at 2-6°C. RESULTS: At baseline, significant reduced levels of factor V, free protein S, α2-antiplasmin and tPA-induced ROTEM lysis time were observed in Omniplasma as compared to quarantine plasma. Moreover, thrombin generation, IXa-AT complex levels and factor XIa were significantly increased in Omniplasma. The majority of the parameters studied remained stable in Omniplasma 48 h after thawing, with the exception of factor VIII (decrease) and IXa-AT (increase). CONCLUSION: Our results suggest an increased coagulation potential, presumingly as a result of contact activation during the production process and also, an increased fibrinolytic potential in Omniplasma. The stability of Omniplasma, based upon the different parameters studied, is comparable to Q-plasma. A maximum post-thawing time of 48 hfor Omniplasma can be suggested.

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.

Cirrhosis patients have a coagulopathy that is associated with decreased clot formation capacity.

BACKGROUND: The coagulopathy in cirrhosis is associated with thrombosis and bleeding. OBJECTIVES: To gain better insights into the coagulopathy in patients with cirrhosis, we evaluated plasma thrombin generation and whole blood clot formation in a cross-sectional study. METHODS: Blood was collected from 73 patients with all-cause cirrhosis (Child-Pugh-A n = 52, B n = 15, C n = 6) and 20 healthy controls. Activity of the coagulation pathways was measured with assays for factor (F) VIIa and FIXa-antithrombin and FXa-antithrombin complexes, respectively. Thrombin generation by calibrated automated thrombography was determined in platelet-poor plasma using a 1 or 5 pm tissue factor trigger with/without thrombomodulin. ROTEM measurements were performed in whole blood triggered with 35 pm tissue factor without/with 175 ng mL(-1) tissue plasminogen activator (the latter refered to as 'tPA-ROTEM'). RESULTS: We observed an increased generation of FVIIa and a moderately elevated amount of FIXa (in complex with antithrombin) without apparent increase in FX activation in patients with cirrhosis. In accordance with this prothrombotic state, markers of thrombin generation potential were also increased upon increasing severity of cirrhosis. In the whole blood clotting assay we observed delayed clot formation and decreased clot strength associated with increased severity of cirrhosis. No significant differences were found for tPA-ROTEM parameters of clot degradation. CONCLUSION: These results indicate that cirrhosis patients have an overall procoagulant plasma milieu but a decreased whole blood clot formation capacity with an apparently unaltered resistance to clot lysis.

Effect of low-dose supplements of menaquinone-7 (vitamin K2 ) on the stability of oral anticoagulant treatment: dose-response relationship in healthy volunteers.

BACKGROUND AND OBJECTIVE: Despite the worldwide use of vitamin K antagonists (VKAs), there is limited knowledge of the influence of dietary vitamin K on anticoagulation control. In view of the increasing nutraceutical availability of menaquinone-7 (MK-7; vitamin K2 ) and its promotion for bone and cardiovascular health, it is important to determine the posology for the interference of supplemental MK-7 with VKA therapy. PATIENTS: Eighteen healthy men and women were anticoagulated for 4 weeks with acenocoumarol, and 15 of them attained a target International Normalized Ratio (INR) of 2.0. In the six subsequent weeks, subjects were given increasing doses of MK-7 (10, 20 and 45 µg day(-1) ) while continuing acenocoumarol treatment at established individual doses. RESULTS: Apart from the INR, acenocoumarol treatment significantly increased the levels of uncarboxylated factor II (ucFII), uncarboxylated osteocalcin (ucOC), and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP), and decreased endogenous thrombin generation (ETP). A daily intake of 45 µg of MK-7 significantly decreased the group mean values of both the INR and ucFII by ~ 40%. Daily intakes of 10 and 20 µg of MK-7 were independently judged by two hematologists to cause a clinically relevant lowering of the INR in at least 40% and 60% of subjects, respectively, and to significantly increase ETP by ~ 20% and ~ 30%, respectively. Circulating ucOC and dp-ucMGP were not affected by MK-7 intake. CONCLUSIONS: MK-7 supplementation at doses as low as 10 µg (lower than the usual retail dose of 45 µg) significantly influenced anticoagulation sensitivity in some individuals. Hence, the use of MK-7 supplements needs to be avoided in patients receiving VKA therapy.

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.