Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of β3 integrins

Abstract

Extracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial β3 integrin α(V)β(3), the platelet integrin α(IIb)β(3) binds PDI. PDI also binds to recombinant β3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laser-induced arteriolar wall injury is markedly reduced in β3-null (β3(-/-)) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in β3(-/-) mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial α(V)β(3) versus platelet α(IIb)β(3) or α(V)β(3), we performed reciprocal bone marrow transplants on wild-type and β3(-/-) mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with β3(-/-) bone marrow or in β3(-/-) mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet β3 integrins contribute to extracellular PDI binding at the vascular injury site.

Figure 1

PDI interacts with β3 integrins on cell surfaces. (A) Interaction of PDI with P-selectin, α_IIbβ₃, or α_Vβ₃. Histograms show fluorescence intensity of Alexa 488–labeled recombinant PDI interacting with CHO cells expressing either P-selectin (red), α_IIbβ₃ (green), or α_Vβ₃ (blue). CHO cells were pretreated for 10 minutes in PBS with or without Mn²⁺ and incubated with PDI labeled with Alexa 488. (B) CHO cells expressing either α_IIbβ₃ (left) or α_Vβ₃ (right) were incubated with Alexa 488–labeled PDI in the presence of Mn⁺² (green) or EDTA (blue). The interaction of PDI with CHO cells expressing P-selectin (red) is shown as control.

Figure 2

PDI interaction with α_IIbβ₃ and β3 by surface plasmon resonance. Full-length α_IIbβ₃ (25 μg/mL) or recombinant β3 (25 μg/mL) in 10mM acetate buffer, pH 5 or pH 4.5, respectively, was immobilized on the surface of a Biacore CM5 chip. Different concentrations of PDI (21μM [magenta], 7μM [gray], 2.33μM [gold], 0.78μM [turquoise], 0.26μM [pink], 0.09μM [blue], 0.03μM [green], or 0μM [red]) were injected, and the experiments were performed in duplicate with a running buffer of 10mM HEPES, pH 7.5, 150mM NaCl, 0.05% P20, 2mM MnCl₂. The dissociation constant, K_d, was calculated based on the K_on and K_off value. (A) PDI binds to α_IIbβ₃ with a K_d of 0.67μM. (B) PDI binds to β3 with a K_d of 1.2μM.

Figure 3

Platelet thrombus formation after laser-induced arteriolar wall injury in β3^−/− mice. (A) Nonblocking rat anti–mouse GPIbβ antibody conjugated to DyLight 649 (0.2 μg/g body weight) was infused into a wild-type (wt) or β3^−/− mouse 5 minutes before laser-induced arteriolar wall injury. Representative images of fluorescence associated with platelets (red) are shown over the course of 180 seconds after vessel injury within the context of the bright-field microvascular histology. (B) The median integrated platelet fluorescence (F_platelets) for 22-23 thrombi in 2 WT or 2 β3^−/− mice infused with the anti-GPIbβ antibody conjugated to DyLight 649 is presented over the course of 250 seconds after vessel injury.

Figure 4

Comparison of PDI secretion from WT and β3^−/− mouse platelets. (A) Platelets from wild-type (wt) and β3^−/− mice (1 × 10⁹ platelets/100 μL) were incubated with thrombin for 5 minutes, and the supernatant and pellet were separated. The pellet was solubilized with 0.1 mL of SDS lysis buffer. Washed human platelets (1 × 10⁹ platelets/mL) were lysed with 0.1 mL of SDS lysis buffer. Washed human platelets (10 μL), the platelet releasates (S; 5 μL), and platelet lysates (P; 5 μL) of WT and β3^−/− mouse platelets (5 μL) were electrophoresed in an SDS-PAGE gel and immunoblotted with rabbit polyclonal anti-PDI antibodies or nonimmune rabbit IgG. hplt indicates human platelets.

Figure 5

PDI accumulation and platelet thrombus formation in β3^−/− mice. Noninhibitory rabbit anti-PDI antibody conjugated to Alexa 488 (0.5 μg/g body weight) and antifibrin-specific antibody conjugated to Alexa 647 (0.3 μg/g body weight) were infused into a wild-type (wt) or β3^−/− mouse 5 minutes before laser-induced arteriolar wall injury. (A) Representative images of the fluorescence signals associated with PDI (green) and fibrin (red) are shown over a course of 180 seconds after vessel injury within the context of the bright-field microvascular histology. The median integrated fluorescence associated with PDI fluorescence (F_PDI) and fibrin fluorescence (F_fibrin) after infusion of anti-PDI antibodies (B) and antifibrin antibodies (C) in 3 WT mice (n = 28 thrombi) and 3 β3^−/− mice (n = 25 thrombi) is presented over a course of 250 seconds after vessel wall injury.

Figure 6

Analysis of chimeric and homozygous mouse blood. (A) PCR was performed with whole blood of β3^−/−, WT, and WT/β3^−/− and β3^−/−/WT chimeric mice. PCR products were obtained as 538 bp for β3⁻/WT and 446 bp for WT/β3^−/−. (B) Blood cell counts of mice after bone marrow transplantation. Complete blood counts were obtained, including red cells (RBC), platelets (PLT), white blood cells (WBC), neutrophils (NE), lymphocytes (LY), and monocytes (MO). Data represent mean ± SD of 4-5 independent experiments.

Figure 7

PDI and platelet accumulation in chimeric mice after vascular injury. Chimeric mice were generated by reciprocal bone marrow transplantation between wild-type (WT) and β3^−/− mice. Noninhibitory rabbit anti-PDI antibody (0.5 μg/g body weight) conjugated to Alexa 488 was infused into mice with a nonblocking rat anti–mouse GPIbβ antibody conjugated to DyLight 649 5 minutes before laser-induced arteriolar wall injury. Representative images associated with platelets (red) and PDI (green) are shown over a time course of 180 seconds after vessel injury within the context of the bright-field microvascular histology.

Figure 8

Quantitation of fluorescence associated with PDI and platelets in 4 transplanted mouse cohorts. The median integrated platelet (A) and PDI (B) fluorescence (F) after infusion of anti-GPIb antibody and anti-PDI antibody in each of the transplanted mice is presented over a time course of 240 seconds after vessel wall injury. 1 indicates WT/WT (n = 25 thrombi); 2, WT/β3⁻ (n = 28 thrombi); 3, β3⁻/WT (n = 26 thrombi); and 4, β3⁻/β3⁻ (n = 26 thrombi; donor/recipient).