Coagulation on Endothelial Cells: The Underexposed Part of Virchow’s Triad

Abstract

The process of thrombin generation involves numerous plasma proteases and cofactors. Interaction with the vessel wall, in particular endothelial cells (ECs), influences this process but data on this interaction is limited. We evaluated thrombin generation on EA.hy926, human coronary arterial ECs (HCAECs) and patient-derived human venous ECs (HVECs) by means of a modified calibrated automated thrombogram (CAT) method and especially looked into contribution of the intrinsic and extrinsic pathways. Thrombin generation was measured in presence of confluent ECs with normal pooled and factor XII-deficient (FXII-deficient) platelet-poor plasma, with/without active site inhibited factor VIIa (ASIS) to block the extrinsic pathway and corn trypsin inhibitor for blocking contact activation (intrinsic pathway). Fetal bovine serum (FBS) was removed from culture conditions as FXIIa from the serum retained on ECs apparently, thereby inducing strong contact activation. In serum-free conditions, EA.hy926 and patient-derived HVECs induced thrombin generation mainly via the contact activation pathway with minor influence of ASIS on peak height and very low thrombin generation curves in FXII-deficient plasma. HVECs derived from coronary arterial bypass graft (CABG) patients showed increased thrombin generation compared to control patients, which could be ascribed to increased contact activation. Contribution of the extrinsic pathway on patient-derived ECs was limited. We conclude that the CAT method in combination with serum-free cultured ECs offers a valuable high-throughput method to evaluate endothelial influences on thrombin generation, which appears to involve predominantly contact activation on ECs. Contact activation-mediated thrombin generation was increased on ECs from CABG patients compared to controls.

Coagulation Factor XII –A Key Pro-Inflammatory and Pro-Coagulant Protein

“…Although the proinflammatory and pro-coagulation cascade of FXII has been intensively studied in vitro, how it functions in vivo is not so clear. Studies have shown that collagenactivated platelets release large amount of Zn 2+ , and under high concentrations of Zn 2+ , FXII may bind to urokinase-type plasminogen activator receptor (u-PAR), complement protein C 1 q receptor, (gC 1 qR) and corner binding protein-1 on the surface of human umbilical vein endothelial cells (HUVECs) [3,12]. During which, the 40-44 and 78-82 residues of the fibronectin Type II domain on the N-terminal of FXII bind to two Zn…”

Phenotypic Fitness of Primary Endothelial Cells Cultured from Patients with High Cardiovascular Risk or Chronic Kidney Disease for Vascular Tissue Engineering

“…24 In brief, the method employs a low-affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC) to continuously monitor thrombin activity in clotting plasma. Normal plateletpoor plasma was obtained from pooling plasmas of 80-90 healthy donors, according to the standard procedure of the Laboratory of Hematology, Maastricht University Medical Center.…”

“…Then, the cells were washed with HEPES buffer, and a previously described thrombin generation assay was performed. 24 Measurements were conducted in 80 mL human plateletpoor normal-pooled plasma (NP) or FXII-deficient plasma in a total volume of 120 mL. To the 80-mL plasma sample, 20 mL trigger reagent (0 pM tissue factor [TF], 24 mM phospholipids at 20:20:60 mol% [phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine, PS:PE:PC]) was added.…”

“…32 In our analysis, no significant differences were seen, which is possibly related to large variations in the CKD group, as we showed enhanced thrombin generation in CABG-derived HVECs compared with controls in an earlier report. 24 Patients included in the CKD group varied substantially in duration of renal failure as well as in patient characteristics such as age and comorbidities. Several studies have reported a relationship between thrombin generation in plasma and CAD, 33,34 although results are not very consistent.…”

Incidence of and risk factors for persistent gram-positive bacteraemia and catheter-related thrombosis in haematopoietic stem cell transplantation

“…2,3 The pathogenesis of CVC-related thrombosis is complex and is thought to result from activation of coagulation pathways by the foreign material in the bloodstream with formation of a thrombin sheath covering the CVC, the occurrence of vascular endothelial damage and endothelial cell activation by disturbed haemodynamics. 4 Infection can also stimulate thrombus formation by aggravating coagulation, but the presence of a thrombus mass or thrombin sheath around the CVC also increases the risk for microbial colonisation and bacteraemia. 5 The CVC-related thrombosis can result in serious complications, including pulmonary embolism and, when infected, it increases the risk of endocarditis and septic embolisms considerably.…”