Evaluation of von Willebrand factor and von Willebrand factor propeptide in models of vascular endothelial cell activation, perturbation, and/or injury

Abstract

Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ∼33% while in control rats VWF increased ∼5%. In dogs, VWF levels transiently increased ∼30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.

Keywords: DDAVP.; DIVI; LPS; VWF; VWFpp; activation; biomarker; dog; endothelial cell; perturbation; rat.

Figure 1

Plasma von Willebrand Factor (VWF) levels with repeat tail vein blood collection in näive rats. Blood was collected from the tail vein at 0, 2, 4, 6, and 24 hr into sodium citrate anticoagulated vacutainers. Samples were centrifuged to obtain platelet poor plasma. Plasma VWF levels were determined using the STA Compact^®. Plotted is the average fold difference from 0 hr with standard error. N = 8 rats. * p < 0.05

Figure 2

Canine plasma VWF levels from multiple venipunctures within a day and over multiple days. Blood was collected from the jugular vein at 0, 1, 2, 3, 5, 24, 48, 72, and 96 hr into 3.8% sodium citrate anticoagulated vacutainers. Samples were centrifuged to obtain platelet poor plasma. Plasma VWF levels were determined using the STA Compact^®. Plotted is the average fold difference from 0 hr with standard error. N = 5 dogs. No statistical differences at any time

Figure 3

Platelet to plasma VWF ratio in rat and dog. Platelet-free plasma (plasma) and lysed platelets in saline (platelets) were evaluated for VWF on a STA Compact^®. N = 11 and 3 for rat and dog, respectively. Plotted are the average and standard error. Note: Rat platelets contain much more VWF than dog platelets, compared to the respective plasma levels.

Figure 4

Plasma VWF levels in control or fenoldopam (FD)-treated double venous cannulated rats. Double cannulated (jugular vein for dosing and femoral vein for blood collection) rats were treated with vehicle or 100 mg/kg/min FD for 24 hr with blood collected 0 (pre-dose), 0.75, 2, 4, 6, and 24-hr postdose initiation. Sodium citrate antic-oagulated blood was centrifuged to obtain platelet poor plasma. Plasma von Willebrand Factor (VWF) levels were determined using the STA Compact^®. Plotted is the average fold difference from 0 hr with standard error. N = 5 per group. * p < 0.05, ** p < 0.01

Figure 5

Evaluation of plasma von Willebrand Factor (VWF) as a drug-induced vascular injury (DIVI) biomarker. Dogs were treated with 15, 60, or 240 mg/kg ZD6169, a PCO, with blood collected 0- (predose), 1.5-, 3-, 6-, and 24-hr postdose. Sodium citrate anticoagulated blood was centrifuged to obtain platelet poor plasma with VWF analyzed using the STA Compact¹. Plotted is the average fold difference from 0 hr with standard error. Heart rate (HR) and mean arterial blood pressure (MAP) were recorded 1-hr postdose. Dogs were euthanatized 24-hr postdose, and hearts were processed, fixed, sectioned, and hematoxylin and eosin (H&E) stained. N = 2 dogs per group.

Figure 6

von Willebrand Factor (VWF) immunohistochemistry staining of canine coronary artery 24 hr after treatment with the PCO ZD6169. Dogs were euthanatized 24 hr after bolus treatment with 60 mg/kg ZD6169. Heart was processed, fixed, sectioned, and immunohistochemically stained for VWF. Note: VWF within the smooth muscle layer (@) of the vascular wall. * is showing endothelial cells expressing VWF.

Figure 7

Plasma VWF, VWFpp, and VWFpp:VWF ratio levels as biomarkers of canine endothelial perturbation. Blood was collected from the jugular vein at predose (0 hr) and 0.25, 1, 3, 6, 12, and 24 hr after dosing with vehicle, 5 μg/kg DDAVP (physiological), and 2 mg/kg endotoxin (pathological). Sodium citrate anticoagulated blood was centrifuged to obtain platelet poor plasma with VWF analyzed using the STA Compact^®. Plotted is the average fold difference from 0 hr with standard error. N = 3 dogs per group. Statistics was not done because of blood unavailability from all dogs at all time points.

Figure 8

Canine plasma VWF, VWFpp, and VWFpp:VWF ratio as DIVI biomarkers. (A) VWF; (B) VWFpp, and (C) VWFpp:VWF. Dogs were treated with 0, 3.6, 10.8, or 27 mg/kg dopamine with blood collected 0- (predose), 1-, 6-, 12-, and 24-hr postdose. Sodium citrate anticoagulated blood was centrifuged to obtain platelet poor plasma and VWF (0, 1, and 6 hr only) analyzed using the STA Compact^®. VWFpp was analyzed by the enzyme-linked immunosorbent assay (ELISA). Plotted are the mean and standard error values normalized to predose. N = 5 dogs per group. *p < .05. **p < .01.