Valves of the Deep Venous System: The Overlooked Risk Factor.
Authors: Brooks Erin , Wadsworth Marilyn , Taatjes Douglas , Trotman Winifred , Evans Mark , Ittleman Frank , Callas Peter , Esmon Charles , Bovill Edwin 
Published: April 2020

Abstract
The valves of the deep venous system were identified as major sites of initiation of deep venous thrombosis in the 1950s. Stasis in the valves has been associated with increased hematocrit, lower PO2 and the presence of local eddy currents. However, the contribution of venous endothelium to thrombosis risk has received little attention. In recent years several publications have emphasized the importance of endothelial heterogeneity in different vascular beds. We hypothesized that the endothelium of the valve sinuses would differ from the non-valvular venous wall with up regulation of anticoagulant and down regulation of procoagulant activities, thus acting as a deterrent to venous thrombosis. In pursuit of this hypothesis we measured by fluorescence confocal microscopy the endothelial protein C receptor (EPCR), thrombomodulin (TM) and von Willebrand Factor (VWF) in saphenous veins obtained from cardiac bypass surgery (CABG). Saphenous veins (5 specimens, 9 valves) from CABG patients were obtained in the operating room and fixed with 10% neutral buffered formalin for 24 hours. Representative sections were taken from valvular and non-valvular venous wall. Slides were prepared for confocal microscopy (Zeiss LSM 510 META) from paraffin embedded tissue. The following antibodies were used: Primary (TM-mouse mAB (clone 141C01) Lab Vision, EPCR-goat polyclonal R&D Systems and VWF-rabbit polyclonal DAKO): Secondary (Alexa α-IgG: 647 donkey α-mouse, 555 donkey α-goat and 488 donkey α-rabbit). The slides were imaged with a 25X oil immersion lens and then 150μM lengths of vessel wall were demarcated and the fluorescence intensity of antibody binding was measured as Arbitrary Intensity Units (AIU) using MetaMorph image analysis software. Measurements were taken from a representative section of venous vessel wall just distal to the valve and from two adjacent segments at the junction of the valve leaflet and venous wall at the bottom of the valve sinus. Repeated measures ANOVA was used to compare the fluorescence intensity of antibody binding between areas for each protein. Pairwise comparisons were conducted for significant ANOVAs. TM (p=0.016) and EPCR (apparent trend) appear to be increased in the valve sinuses compared to the non-valvular venous wall and VWF (p=0.003) appears to be decreased in the valve sinuses compared to the non-valvular wall. In addition there is notable inter-individual variation in the expression of these proteins. These preliminary data suggest that the procoagulant/anticoagulant balance differs significantly between the valvular and non-valvular venous wall with the venous sinus shifted to a thromboresistant phenotype. Variation in venous sinus thrombo-resistance may be an important factor in venous thrombogenesis. Further studies of this overlooked risk factor appear to be warranted.
Comparisons of non-valvular venous wall to valvular sinus endothelium: Arbitrary Intensity Unit (AIU) Location [AIU Mean (SEM)] Protein Vein Wall Valve Wall Valve Wall ANOVA p value Significant Differences VWF 1,955,265 (613,096) 178,229 (82,545) 553,864 (167,070) 0.003 1 &gt; 3 &gt; 2 EPCR 685,210 (345,153) 1,581,627 (377,537) 1,791,428 (362,861) 0.095 NONE TM 747,340 (270,145) 2,754,752 (788,827) 2,459,144 (477,396) 0.016 1 &lt; 2 = 3