Contribution of platelet vs. endothelial VWF to platelet adhesion and hemostasis

Abstract

Background: von Willebrand factor (VWF) is a glycoprotein that plays an important role in primary hemostasis. VWF is synthesized and stored in endothelial cells (ECs) and megakaryocytes/platelets. Plasma VWF is primarily derived from ECs and is generally believed to be essential for hemostasis. VWF synthesized in megakaryocytes is stored in platelet α-granules, from which it is released following platelet activation. The relative contribution of VWF stored in ECs or megakaryocytes/platelets or present in plasma to hemostasis is not clear.

Objectives: We investigated whether EC-derived VWF plays the major role in hemostasis while the contribution of platelet-derived VWF is negligible, or if platelet-derived VWF also significantly contributes to hemostasis.

Methods and results: Mice expressing VWF only in ECs (EC-VWF) or platelets (Plt-VWF) were created by reciprocal bone marrow transplantation between C57BL/6J (WT) and VWF knockout mice (VWF-/-). Plasma VWF levels in EC-VWF were similar to WT. Plt-VWF mice had a trace amount of VWF in their plasma while VWF levels in platelet lysate were comparable to WT. Tail bleeding time was normal in EC-VWF. Interestingly, Plt-VWF showed partially corrected bleeding time and significantly decreased blood loss volume compared with VWF-/-. Adhesion of platelets perfused over immobilized collagen under shear stress was significantly higher in both EC-VWF and Plt-VWF compared with VWF-/-.

Conclusion: VWF synthesized in ECs is sufficient to support hemostasis in VWF-/- mice, and VWF produced in megakaryocytes/platelets can also contribute to hemostasis in the absence of EC-derived VWF.

Fig. 1

Murine models of EC-VWF and Plt-VWF established by crossed BMT. (A) Plasma samples and (B) platelet lysates collected from WT, VWF^−/−, EC-VWF, and Plt-VWF mice were analyzed by ELISAto measure VWF antigen levels. EC-VWF mice had WT levels of plasma VWF, but no platelet VWF. Plt-VWF mice had WT levels of platelet VWF and a trace amount of plasma VWF. (C) FVIII activity in mouse plasma was quantified by chromogenic assay. FVIII:C of EC-VWF mice were similar to WT mice. Plt-VWF mouse plasma presented low FVIII:C but the levels were significantly higher than VWF^−/− mice. NS, not statistically significant, *P < 0.05

Fig. 2

Platelet adhesion on type I collagen under flow. (A) Platelets in mouse whole blood from WT, VWF^−/−, EC-VWF, and Plt-VWF were labeled with mepacrine and perfused over Vena8Fluor+Biochips coated with type I collagen at a shear rate of 2,000 s⁻¹. Images were taken after 120 seconds of perfusion. (B) Percent of surface area covered by platelets after 120 seconds of perfusion was calculated using Metamorph software and plotted for each group of mice. Platelet adhesion of EC-VWF mouse blood samples to type I collagen were similar to WT mice. Surface coverage of Plt-VWF mouse blood samples was significantly higher than VWF^−/− mice. *P < 0.05, **P < 0.001

Fig. 3

Tail bleeding assays. (A) WT, VWF^−/−, EC-VWF, and Plt-VWF were analyzed by tail bleeding time assays 10 weeks after transplantation. (B) The amount of blood lost into collection tubes was determined by measuring hemoglobin content. Bleeding time and blood loss volume were normal in EC-VWF. Plt-VWF had a partially corrected bleeding time and significantly reduced blood loss volume compared to VWF^−/−. *P < 0.05

Fig. 4

Epinephrine-stimulated release of VWF. Epinephrine was subcutaneously administered to animals. Plasma samples were collected before and 30 minutes after injection. VWF antigen levels were determined by ELISA. Plasma VWF levels were significantly increased by epinephrine infusion in EC-VWF mice and WT mice but not in Plt-VWF mice. NS, not significant, *P < 0.05