A local increase in red blood cell aggregation can trigger deep vein thrombosis: evidence based on quantitative cellular ultrasound imaging

Abstract

Background: Recurrent deep vein thrombosis (DVT) risk factors include a first idiopathic DVT, strongly suggesting the existence of undiagnosed and/or unidentified prothrombotic abnormalities.

Objectives: To evaluate the impact of locally increased red blood cell (RBC) aggregation on DVT pathogenesis in a rabbit model.

Methods: DVT presence, flow and aggregation were measured in situ with ultrasound. Greatly enhanced aggregation was achieved by covalent linkage of Pluronic F98 to the RBC surface; coating with Pluronic F68, which very mildly enhances aggregation, was used as a coating control. On day 1, endothelial damage and a partial stenosis were surgically created on the left femoral vein whereas the right femoral vein was not manipulated.

Results: A thrombus was formed within 30 min in six out of seven left femoral veins of animals receiving a 30% volume blood exchange with F98-coated RBC, whereas a thrombus occurred in only one out of seven veins in F68-transfused controls. In vivo imaging using quantitative ultrasound confirmed increased aggregation in the thrombosed veins of the F98 group compared with the F68 group and the contralateral vessel. For each group, five animals were followed for 2 weeks before being killed. In F98-transfused animals, lysis of clots occurred and the presence of chronic thrombi totally occluding the vein in three out of five animals was confirmed by histology. Conversely, in the F68 group, a single disorganized blood clot was observed in one out of five animals.

Conclusions: A marked increase in RBC aggregation promotes thrombosis in rabbit femoral veins, confirming a pathophysiological role of locally altered hemorheology in the onset of DVT.

Figure 1

(a) Low shear viscosity as a function of the shear rate for 30% Pluronic F68-coated RBC, 30% Pluronic F98-coated RBC, and normal values for uncoated rabbit and human bloods taken from [31]. Microscopic images of diluted rabbit RBC (H~5%) suspended in native plasma at 37°C for: (b) normal rabbit blood; (c) 30% Pluronic F68-coated RBC showing RBC aggregates comparable to normal rabbit blood; and (d) 30% Pluronic F98-coated RBC showing enhanced aggregation.

Figure 2

B-mode ultrasound images of the prothrombotic left vein (LFV) and the contralateral control right vein (RFV) for F68 and F98 transfused rabbits. The partial stenosis is indicated by an arrow on the LFV panels. The strong venous echogenicity in F98 transfused animals is indicative of RBC aggregation.

Figure 3

Bright field microscopy of H&E stained cross sections of LFV at day 14 upstream of the silk suture producing the partial stenosis showing: a) neointimal hypersplasia found in the F68 group LFV; b) a disorganized young thrombus that occurred in only 1 of 5 F68 animals; c) and d) completely obstructed lumen by partially organized thrombi in a F98 group rabbit.

Figure 4

B-mode images and superimposed parametrical images of in situ RBC aggregation level determined by the structure parameter D (SFSAE) for RFV and LFV in one F98 rabbit. The D parameter is the mean aggregate diameter in terms of number of RBC [17].

Figure 5

Aggregate size parameter D (defined as the ratio of the diameter of a fractal isotropic aggregate to the diameter of one RBC, that is smaller than 1 for disaggregated RBC [17], at d0, d1, d4, d9 and d14 for the control F68 and hyperaggregating F98 groups. Significant differences (*p < 0.05) were found for D in the F98 group between LFV and RFV on d1, d4 and d9, and between d1 and all other time points within LFV in F98. On d1, significantly different values of D were found between the F68 and F98 groups.