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

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

Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test

<div><p>Introduction</p><p>Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.</p><p>Methods</p><p>Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.</p><p>Results</p><p>Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.</p><p>Conclusions</p><p>The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.</p></div

Effect of temperature on platelet function testing by flow cytometry.

<p>Immediately after blood donation, blood was stored at RT or at 37°C. At each time point (X-axis), platelet activation in response to TRAP (panel a), CRP (panel b) or ADP (panel c) was tested. For the 37°C condition, blood was immediately placed at 37° and platelet function tests were performed at 37°C. For the RT condition, blood was kept at RT and the tests were performed at RT. For the RT/37°C condition, blood was kept at RT and 10 min before performing the platelet function test, blood was placed at 37°C and tests were performed at 37°C. The median fluorescence intensity (MFI) of αIIbβ3 receptor activation (left panels) and P-selectin expression (right panels) was recorded and expressed as the percentage compared to the MFI of the 37°C condition measured after 30 min at 37°C. Data are presented as mean ± SD (n = 3 to 5).</p

Assay variation when blood is stored at RT up till 240 min followed by 10 min preincubation at 37°C prior to performing the platelet function test.

<p>Assay variation when blood is stored at RT up till 240 min followed by 10 min preincubation at 37°C prior to performing the platelet function test.</p