Kallikrein augments the anticoagulant function of the protein C system in thrombin generation

Background Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project. Methods Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. Results Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 x 10(-8)). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor. Conclusions Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis

Increased Clot Formation in the Absence of Increased Thrombin Generation in Patients with Peripheral Arterial Disease: A Case-Control Study

BackgroundIn peripheral arterial disease (PAD), activation of the hemostatic system may contribute to atherosclerosis progression and atherothrombotic events.ObjectiveThis case–control study assesses the overall coagulation status in PAD patients by evaluating coagulation markers in combination with thrombin generation potential, whole blood (WB) clot formation, and fibrinolysis.MethodsIn blood from 40 PAD patients (n = 20 with cardiovascular event within 1 year after initial diagnosis, n = 20 without) and 40 apparently healthy controls, thrombin generation was determined in WB and platelet-poor plasma. Whole blood rotational thromboelastometry (ROTEM) measurements were triggered with tissue factor with/without tissue plasminogen activator.ResultsWe observed increased levels of erythrocyte sedimentation rate, leukocytes, eosinophil granulocytes, vWF antigen, fibrinogen, and D-dimer in PAD patients (p < 0.05). Markers of thrombin generation potential showed no difference between patients and healthy controls. In PAD patients with event compared to patients without, WB-thrombin generation showed a lower thrombin potential when triggered with 0 and 2.5 pM tissue factor. The ROTEM clotting assay showed significantly faster clot formation and increased clot firmness in PAD patients compared to controls. No significant differences were found for parameters of clot degradation.ConclusionThere are no significant differences between the thrombin generation profiles of PAD patients and healthy controls. Between PAD patients with and without cardiovascular event, the WB thrombin generation appears to differ. Mechanistically, PAD patients show an increased ability to form a stable clot in WB in comparison to healthy controls. This is most likely due to the increased fibrinogen levels related to the inflammation in atherosclerosis, confirming the importance of the inflammation-coagulation axis

Activation of the contact system in patients with a first acute myocardial infarction

AbstractIntroductionThe contribution of the contact system to arterial thrombosis is unclear, results of clinical studies are conflicting. Particularly, little is known about the involvement of the contact system in the progression of arterial thrombosis. Therefore, we investigated the activation of the contact system during an acute myocardial infarction (AMI) and 3 and 6months following the acute event.MethodsPlasma of patients with a first AMI was collected on admission and 3 and 6months after the AMI. The levels of complexes of activated factor XI (FXIa), FXIIa and kallikrein with C1 esterase inhibitor (C1INH) and the levels of complexes of FXIa with α1-antitrypsin (AT) were measured in these plasmas. Recurrent cardiovascular events were recorded during a one year period after the AMI.ResultsWe observed that the levels of FXIa-C1INH were elevated during the acute phase compared to the steady-phase 3 and 6months after the AMI. The levels of FXIa-AT, FXIIa-C1INH and kallikrein-C1INH did not change over time. The levels of FXIa-C1INH, FXIa-AT, FXIIa-C1INH and kallikrein-C1INH were not predictive for a recurrent event.ConclusionWe observed that during an AMI, the activation of FXI was increased. The levels of FXIIa-C1INH were not elevated, suggesting that activation of FXI during the acute phase did not result from contact activation. The levels of the enzyme inhibitor complexes were not predictive for a recurrent event one year after the first AMI

Different homing pattern of isolated mouse lymphoma cells correlates with a different chromosomal pattern

After 5–20 weeks of in vitro culture of mouse lymphoma cells, a characteristic and reproducible change in cell morphology, clonogenic ability, and homing pattern after intraperitoneal or intravenous injection was observed. Cytogenetic comparison of the two cell populations present before and after the “switch” revealed that the phenotypic changes cannot be due to in vitro karyotype evolution because their chromosomal pattern differed in such a way that it is impossible that they can evolve from each other. It was concluded that two different cell populations are present in the lymphoma and their growth and behavior are influenced by certain circumstances and/or interactions. Apparently one population predominates in the peripheral blood circulation, whereas the other will predominate after prolonged in vitro culturin

The effects of oral contraceptive usage on thrombin generation and activated protein C resistance in Saudi women, with a possible impact of the body mass index

BACKGROUND AND OBJECTIVES:The effect of oral contraceptive (OC) usage on coagulation has been studied worldwide. However, no such studies have been conducted in Saudi Arabia on Saudi women using OCs. The aim of this study was to investigate the effects of OC-induced changes of thrombin generation (TG) in the absence and presence of activated protein C (APC) or thrombomodulin (TM) in Saudi women. METHODS:A total of 115 adult women, 47 on oral contraception (OC users) and 68 controls (not using OCs) were recruited from the obstetrics-gynecology outpatient clinic in Saudi Arabia. OCs that were used in this study include the following: Marvelon, Gynera, Cerrazetem, Yasmine, Microlut, Gracial and Diane. The plasma calibrated automated thrombinography (CAT) was used to determine TG which was expressed as endogenous thrombin potential (ETP; nM/min), lag time (min), peak (nM) and time-to-peak (ttpeak; min). In the presence of TM or APC, TG parameters were expressed relative to the parameters in the absence of TM or APC. RESULTS AND CONCLUSION:As in other populations, our study demonstrated that OC usage induced prothrombotic changes in plasma of Saudi women, including resistance to the inhibitory actions of TM and APC. More specifically, OC usage in our population predominantly influenced TG and APC/TM sensitivity in overweight and obese individuals, a finding that needs confirmation in large cohort studies. The effects of APC and TM on TG parameters showed a positive association, and the correlation coefficients were higher in OC users for both ETP and peak values

Data_Sheet_1_Assessing the individual roles of FII, FV, and FX activity in the thrombin generation process.PDF

Thrombin generation (TG) is known as a physiological approach to assess the hemostatic function. Although it correlates well with thrombosis and bleeding, in the current setup it is not sensitive to the effects of fluctuations in single coagulation factors. We optimized the calibrated automated thrombinography (CAT) method to quantify FII, FV and FX activity within the coagulation system. The CAT assay was fine-tuned for the assessment of FII, FV and FX by diluting the samples in FII-, FV-, or FX-deficient plasma, respectively, and measuring TG. Plasma FII levels correlated linearly with the ETP up to a plasma concentration of 100% FII. FV and FX levels correlated linearly with the peak height up to a plasma level of 2.5% FV and 10% FX, respectively. Sensitized CAT protocols were designed by adding a fixed volume of a pre-diluted patient sample to FII, FV, and FX deficient plasma in TG experiments. This approach makes the TG measurement dependent on the activity of the respective coagulation factor. The ETP or peak height were quantified as readouts for the coagulation factor activity. The intra- and inter-assay variation coefficients varied from 5.0 to 8.6%, and from 3.5 to 5.9%, respectively. Reference values were determined in 120 healthy subjects and the assays were clinically validated in 60 patients undergoing coronary artery bypass grafting (CABG). The sensitized CAT assays revealed that the contribution of FII, FV, and FX to the TG process was reduced after CABG surgery, leading to reduced prothrombin conversion and subsequently, lower TG.</p

Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile

BACKGROUND: Multiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis. OBJECTIVES: To get better insight in the disbalanced hemostasis of MM patients. METHODS: We conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls. RESULTS: In WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls. CONCLUSION: Patients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients