Quiescent platelets stimulate angiogenesis and diabetic wound repair

Abstract

Introduction: Platelets partake in hemostasis, wound healing, and tumor growth. Although platelet-rich-plasma (PRP) has been used in surgery for several years, its mechanism of action and application methods are still poorly characterized.

Materials and methods: A single unit of human platelets obtained by plateletpheresis was diluted in plasma and divided into three equal volumes. One volume was stored at room temperature as fresh platelets (RT), another volume was frozen by storage at -80 degrees C (FZ), and the third volume was frozen at -80 degrees C with 6% DMSO (FZ6). Plasma (PL) was used as control. Using flow cytometry, platelets were tested for platelet glycoprotein GPIb and annexin V binding, as survival and activation markers, respectively. Hemostatic function was assessed by thromboelastometry. In vivo, platelets were topically applied on 1 cm,(2) full-thickness wounds on db/db mice (n = 10/group) and healing was staged microscopically and macroscopically.

Results: All platelet preparations showed hemostatic ability. RT platelets were GPIb positive (nonactivated-quiescent platelets) and stimulated angiogenesis by threefold, and cell proliferation by fourfold in vivo. FZ platelets were positive for annexin V, indicating activated platelets and, in vivo, increased only wound granulation. FZ6 platelets contained 30% nonactivated-quiescent and 50% activated platelets and stimulated granulation, angiogenesis, cell proliferation, and promoted re-epithelialization in vivo.

Conclusions: Platelets showed distinct mechanisms to induce hemostasis and wound healing. Quiescent platelets are required to induce angiogenesis in vivo. Platelets stored at room temperature and frozen with 6% DMSO and stored at -80 degrees C achieved optimal wound healing in diabetic mice.

Copyright 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1. Effects of platelet preservation on platelet GPIb and Annexin V binding

a. RT showed a homogenous population positive for GPIb and negative for annexin V binding. b. FZ had 80% annexin V positive platelets, minimal number of GPIb positive platelets and 20% platelet microparticles after thawing c. FZ6 had a bimodal population with 33% GPIb positive platelets and 50% annexin V positive platelets and only 9% platelet microparticles. RT=Liquid preserved platelets stored at room temperature for 2 days; FZ=platelets frozen without DMSO at −80 C after thawing; FZ6=platelets frozen with 6% DMSO at −80 C after thawing.

Figure 2. Effects of platelet preservation and the concentration of platelets on the R time measured in the thromboelastogram

a. RT had an R value of 13.6 minutes. b. The concentration of RT to 1×109 platelets per ml reduced the R value from 13.6 minutes to 5 minutes. c,d. FZ and FZ6 had R values of 7.3 and 7.6 minutes.

Figure 3. Effect of plasma and platelet preparations on wound healing

100μl of a sample containing 1×109 platelets per ml or plasma were applied in single dose on day 0. a. Plasma alone did not stimulate wound closure, while RT stimulated healing. FZ6 induced greater stimulation of wound closure compared to plasma, RT and FZ. b. The platelet products produced an increase in wound closure compared to plasma. FZ6 produced the highest level of wound closure on day 7. c. RT produced a two-fold increase of contraction on day 7 compared to the other treatments. d. FZ6 produced a 2-fold increase in wound re-epithelialization compared to plasma on day 7. Scale Bar 5mm. Results are expressed as mean ± SD. *=p<0.05 compared to PL. RT=Liquid preserved platelets stored at room temperature for 2 days; FZ=platelets frozen without DMSO at −80 C after thawing; FZ6=platelets frozen with 6% DMSO at −80 C after thawing; PL=plasma.

Figure 4. Effect of plasma and platelet preparations on granulation tissue formation

a. 100μl of the sample contains 1 × 109 platelets per ml FZ6 and FZ increased the thickness of the granulation tissue by 2.5 and 1.9 times, respectively, when compared to plasma alone. The area of granulation tissue also increased 1.9 and 1.6 times following treatment with FZ6 and FZ b,c. Quantification of the results. Results are expressed as mean ± SD. *=p<0.05, compared to PL.

Figure 5. Effect of plasma and platelet preparations on wound vascularity

a. 100μl of sample containing 1×109 platelets per ml FZ6 and RT induced an increase in wound vascularity compared to PL alone, as assessed by the panendothelial marker (PECAM-1) b. A 2.9 and 2.8 increase in wound vascularity was induced by FZ6 and RT compared to PL. Results are expressed as mean ± SD. *=p<0.05 compared to PL.

Figure 6. Effect of plasma and platelet preparations on cell proliferation

a. 100μl of sample containing 1×109 platelets per ml FZ6 and RT increased cell proliferation assessed by Ki 67 positively compared to plasma alone. b. Quantification of the results. Results are expressed as mean ± SD. *=p<0.05 compared to PL.

Figure 7. Wound healing staging system

Immunohistochemical results are plotted to obtain a two-dimensional wound staging system for healing on day 7. Vascularity was assessed by measurement of PECAM-1, a pan-endothelial marker and cell proliferation was assessed by measurement of Ki-67, a marker for cell proliferation. Three distinct wound healing profiles were noted: plasma showed the lower stimulation of both parameters (arbitrary set to 1), RT and FZ6 stimulated both parameters in a similar manner, while results from FZ fell in between these two groups. Results are expressed as mean ± SD. *=p