Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation

Abstract

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

Figure 1

Human CD39 (hCD39) is widely expressed in transgenic mice. (A) Flow-cytometric analysis of leukocytes and platelets from WT (purple filled curve), heterozygote (green line), and homozygote (pink line) transgenic mice. (B) Immunohistochemical analysis (magnification ∞40) and (C) Western blot demonstrating abundant hCD39 expression in tissues of transgenic (CD39) mice but not control (WT) mice. Strong expression was detected on the vascular endothelium (arrows) and on pancreatic islets (arrowhead). (D) Increased CD39 activity in lysates of lung and heart, hand-picked islets, and washed platelets from transgenic mice were compared and expressed as the ratio to the levels in the corresponding tissues in WT mice. Data represents mean ± SEM.

Figure 2

Expression of hCD39 in transgenic mice affects hemostasis and platelet function. (A) Platelet aggregation studies. The presence of hCD39 on the surface of platelets altered the responsiveness of platelets to agonists ADP and collagen. The initial response to both ADP and collagen was attenuated; however, a complete aggregatory response was subsequently demonstrated. Data shown are representative of three different experiments. (B) Tail-bleeding times. Expression of hCD39 significantly prolonged time to hemostasis compared with WT mice either untreated or treated with apyrase. Treatment with apyrase prolonged bleeding compared with WT control. (C) Tail-bleeding times in the adoptive transfer experiments. The presence of hCD39 either on the endothelium or blood components alone was sufficient to induce a bleeding diathesis. Experiments using transgenic mice were terminated (*) at 15 minutes to prevent hemorrhagic death. Data represent mean ± SEM for six mice in each group. IRR, irradiated. (D) Whole blood aggregation. In response to collagen, WT blood (solid line) aggregated normally. This response was completely abolished in whole blood from mice transgenic for hCD39 (broken line).

Figure 3

Expression of hCD39 protects transgenic mice from death due to induced thromboembolism. (A) Survival of WT mice (white bars) and transgenic mice (gray bars) after intravenous injection with ADP, collagen, or both agents. Between five and 14 mice were examined in each treatment group and the three mice in the controls that received saline. The survival advantage afforded by hCD39 was statistically significant for collagen alone and coadministration of ADP and collagen. (B) Histological examination of the lungs from mice treated with ADP and collagen demonstrating occlusion of small vessels (arrows) in the WT but not in the transgenic lung (magnification ∞40).

Figure 4

Expression of hCD39 elevates plasma adenosine and AMP levels. (A) The level of AMP is significantly higher (*P < 0.01 compared with basal) in both WT and hCD39 transgenic mice following challenge with collagen (gray bars) as compared with basal levels (white bars), but the increase in transgenic mice is significantly greater than that observed in the WT mice. (B) Basal (white bars) adenosine levels are comparable between WT and transgenic mice, but following collagen injection (gray bars) the level of adenosine in the transgenic mice is significantly higher.

Figure 5

Expression of hCD39 protects hearts from rejection in an allograft model of cardiac xenotransplantation. Immunohistochemical analysis of grafts removed at 24 hours after Ab injection, demonstrating preserved morphology and minimal platelet deposition in capillaries (arrow) in transgenic grafts. In contrast, WT grafts show a heavy platelet infiltrate (arrow) and destruction of myocardial architecture (magnification ∞20).