In vivo imaging analysis of the interaction between unusually large von Willebrand factor multimers and platelets on the surface of vascular wall

Abstract

To elucidate how unusually large von Willebrand factor (UL-VWF) multimers facilitate thrombus formation, their behavior was analyzed together with that of platelets in living mice deficient in the gene encoding the protease that cleaves UL-VWF, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13-/-). By crossing ADAMTS13-/- mice with green fluorescent protein-expressing transgenic mice (GFP mice), GFP-ADAMTS13-/- mice were obtained. The dynamics of GFP-expressing platelets were monitored employing intravital confocal fluorescent microscopy. Administration of a vasopressin derivative, DDAVP, a secretagogue of VWF increased the number of platelets adhered to vascular endothelial cells (VECs) on mesentery at sites recognized by an anti-VWF antibody. Some of these platelets were interconnected and aligned as beads on a string. They reached their maximum length at 5 min and were longer in GFP-ADAMTS13-/- mice than in GFP mice (5.3 ± 4.3, N = 6 vs 2.9 ± 2.1 μm, N = 4) (mean±SE). Focal injury of VECs by topical application of FeCl(3) developed longer (25, 3-50 vs 10, 2-25 μm, P < 0.01) (mean, 10th-90th percentile) and more stable (1.3, 0.3-6.3 vs 0.3, 0.2-1.3 s, P < 0.01) connected platelets in GFP-ADAMTS13-/- mice than in GFP mice. This study revealed that ADAMTS13 cleaves platelet-bound UL-VWF multimers, both during their secretion from VECs and after their adherence to injured vascular walls in veins. UL-VWF multimers either being secreted from VECs or circulating in plasma of ADAMTS13-/- mice appeared to facilitate the accumulation of longer and more stable VWF strings with more associated platelets on injured vascular walls.

BLOC-2 subunit HPS6 deficiency affects the tubulation and secretion of von Willebrand factor from mouse endothelial cells

“…DDAVP, used to trigger VWF exocytosis in patients (Mannucci, 1997) and in WT (wild-type) mice (Rybaltowski et al., 2011), led to a significant increase in both total and HM VWF by 5 min post-injection, and this increase remained for around 20 min before resolution (Fig. 1A, a and a′).…”

Primary and secondary haemostasis changes related to aging

“…ADAMTS-13 is a plasma metalloprotease that cleaves vWF multimers thereby modulating their participation in primary hemostasis. The insufficient enzymatic activity of ADAMTS-13 causes that vWF is keep in uncleaved high molecular weight form with an increased tendency to thrombosis [42][43][44]. In addition, the increased vWF levels are associated with a proinflammatory state and a decrease in the ADAMTS-13 activity [12,[45][46][47].…”

Unwinding the von Willebrand factor strings puzzle

“…46,47 In vivo, it is debatable whether extraordinarily long VWF strings actually form either through self-association, or by association of plasma multimers with VWF strings, as the length of the strings visualized in vivo range from 20 to 100 m and VWF networks are not generally observed. 42,49 Which factors regulate platelet binding to VWF strings?…”

“…46,47 In vivo, it is debatable whether extraordinarily long VWF strings actually form either through self-association, or by association of plasma multimers with VWF strings, as the length of the strings visualized in vivo range from 20 to 100 m and VWF networks are not generally observed. 42,49 Which factors regulate platelet binding to VWF strings?A remarkable feature of VWF multimers anchored to endothelial cells is that they can rapidly recruit platelets from the circulation, This binding does not result in a rolling movement of the platelets but in a firm adhesion to VWF strings. Bound platelets become activated as evidenced by the presence of P-selectin and activated ␣IIb␤3 on their surface.…”