Platelet PECAM-1 inhibits thrombus formation in vivo

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Figure 1.

Thrombi are larger in PECAM-1–deficient mice. (A) Median thrombus integrated fluorescence (anti-CD41) (Ai) and area (μm²) (Aii) were calculated for thrombi formed in control (+/+) and PECAM-1–deficient (–/–) mice and are plotted against time for a duration of 120 seconds following thrombus initiation (control: n = 14 thrombi; PECAM-1–deficient: n = 20 thrombi). (B) As a measure of thrombus stability, the durations for which thrombi remained above 50% of maximal integrated fluorescence (anti-CD41) (Bi) and thrombus area (Bii) were calculated, and mean values ± SEM are shown. (Bi) *P = .04; (Bii) *P = .02.

Figure 2.

Imaging of thrombus formation in control and PECAM-1–deficient platelets. (A) Control and (B) PECAM-1–deficient mice were infused with Alexa-660–labeled anti-CD41 antibody Fab fragments before thrombus induction in cremaster muscle arterioles using a nitrogen dye laser. Thrombus formation was studied over a period of 3 minutes using a combination of widefield fluorescence and brightfield microscopy. Arrows indicate the direction of blood flow and blue arrowheads indicate the position of laser-induced endothelial damage. Videos representing the formation of thrombi shown in this figure are available as supplemental Videos S1 and S2.

Figure 3.

The contributions of platelet and endothelial cell PECAM-1 to the regulation of thrombus formation. (A) Chimeric mice were generated by bone marrow transplantation and, following recovery, successful engraftment was verified by flow cytometric analysis to detect the presence or absence of PECAM-1 on lymphocytes from WT mice that received transplants of WT bone marrow (Ai), WT mice that received transplants of PECAM-1–deficient bone marrow (Aii), and PECAM-1–deficient mice that received transplants of WT bone marrow (Aiii). (B) Median anti-CD41 integrated fluorescence (Bi) and thrombus area (μm²) (Bii) were calculated for thrombi formed in chimeric mice generated by bone marrow transplantation and are plotted against time for a duration of 120 seconds following thrombus initiation (Bi: WT mice with WT BM: n = 30, PECAM-1–deficient mice with WT bone marrow: n = 33, WT mice with PECAM-1–deficient bone marrow: n = 14; ii: WT mice with WT BM: n = 26, PECAM-1–deficient mice with WT bone marrow: n = 33, WT mice with PECAM-1–deficient bone marrow: n = 14). KO indicates knock-out. (C) Fibrin deposition in thrombi was measured simultaneously with platelet accumulation for each type of mouse chimera that received transplants and is plotted against time for the duration of 120 seconds following thrombus initiation (WT mice with WT BM: n = 30; PECAM-1–deficient mice with WT bone marrow: n = 32; WT mice with PECAM-1–deficient bone marrow: n = 14).

Figure 4.

Quantitative analysis of ferric chloride (FeCl₃)–induced thrombus formation in the carotid arteries of PECAM-1–deficient and wild-type mice. Filter papers saturated with 10% FeCl₃ were applied to exposed carotid arteries for 3 minutes to induce acute injury to the endothelium, after which the vessels were rinsed and saturated with saline solution. Blood flow was monitored with a miniature Ultrasound Doppler flow probe placed under the exposed artery and recorded using a Transonic Model T106 flow meter. The time for blood flow to drop to 25% of baseline values (75% occlusion) was determined. Each data point reflects the time for the left carotid artery to become 75% occluded in PECAM-1–deficient (left) or wild-type (right) animals. The solid line through each data set represents the mean time to 75% occlusion ± standard deviations. The mean time to 75% occlusion (± standard deviation) for PECAM-1–deficient mice was 8.1 ± 1.1 minutes (n = 12), which was significantly shorter (P < .03) than that observed in wild-type mice (10.0 ± 2.7 minutes, n = 14).