Mice lacking the extracellular matrix protein MAGP1 display delayed thrombotic occlusion following vessel injury

Abstract

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis.

Figure 1

Effect of MAGP1 deficiency on thrombotic occlusion of the carotid artery. (A) Blood flow in the common carotid artery was monitored continuously with an ultrasonic flow probe. Local endothelial injury was induced by application of a 540-nm laser beam to the carotid artery followed by injection of rose bengal dye (50 mg/kg) into the lateral tail vein. Shown is the time to occlusion of blood flow following injury. Error bars indicate mean plus or minus standard deviation (n = 8 for each group). **P < .005, *P < .05. (B) Representative blood flow recordings showing the delayed occlusion time and stochastic flow pattern in MAGP1^−/− animals. Rose bengal dye was injected at time = 0 minutes. (C) Infusion of recombinant MAGP1 re-establishes normal occlusion time in MAGP1^−/− mice. Recombinant bovine MAGP1 was injected into the tail vein as a single bolus 5 minutes before rose bengal injection. An equivalent bolus of saline served as the control. Error bars indicate mean plus or minus SD (n = 6 for each group).

Figure 2

Immunohistochemistry showing localization of infused, recombinant bovine MAGP1 at the site of vascular injury. (A) Photomicrographs on the left are cross sections of carotid arteries from MAGP1^+/+ and MAGP1^−/− mice infused with 50 μg/kg recombinant bovine MAGP1 5 minutes prior to laser-induced injury. Vessels were harvested after complete cessation of blood flow and frozen sections immunostained using a bovine MAGP1-specific antibody. Staining is evident in the thrombus of both genotypes but is particularly prominent in the internal elastic lamina in the MAGP1^−/− mouse (↗, and at higher power in panel B). Panels on the right show staining with antibovine MAGP1 of injured vessels from animals not injected with bovine MAGP1.

Figure 3

Platelet function in MAGP1-deficient mice. (A) The percentage of platelets that aggregate in response to different levels of collagen is the same for both genotypes. (B) The presence of MAGP1 (50 μg/mL) has no effect on human platelet aggregation induced by various agonists, including collagen (Col, 10 μg/mL), adenosine 5′-diphosphate (ADP, 20 μM), arachidonic acid (AA, 500 μg/mL), and epinephrine (Epi, 300 μM). Aggregation was monitored by measuring light transmission through a suspension of stirred washed platelets (1-3 × 10⁸/mL for mouse and 2 × 10⁸/mL for human) using an aggregometer. Data are expressed as either the slope of the aggregation curve or as percentage of cells that underwent aggregation. (C) Recombinant bovine MAGP1 has no effect on the ristocetin cofactor activity of human plasma. All experiments contained normal human plasma diluted 1:1 with Tris-buffered saline, yielding 50% ristocetin cofactor activity in the control sample. Error bars indicate mean plus or minus standard deviation of 4 experiments. None of the values differed significantly (P ≥ .25).

Figure 4

Immunoblot of platelet extracts. Bovine washed platelets were boiled in SDS sample buffer and subjected to SDS-PAGE under reducing and nonreducing conditions. Protein bands were visualized by Coomassie blue staining or transferred to nitrocellulose for immunodetection with an antibody to bovine MAGP1. (A) Coomassie blue–stained gel. (B) Immunoblot analysis of proteins in panel A after transfer to nitrocellulose. Lane 1: Platelet extract under nonreducing conditions (no DTT). Lane 2: Platelet extract under reducing conditions (+ DTT). Lane 3: Semipurified bovine MAGP1 expressed by mammalian SaOS2 cells (+ DTT).

Figure 5

Outer diameter versus pressure for the right carotid artery in wild-type and MAGP^−/− mice. Pressure-diameter curve showing that the carotid artery in wild-type (—) and MAGP^−/− (----) animals has identical mechanical properties, identical diameters, and equal pressures.

Figure 6

Analysis of MAGP1 interaction with selected plasma proteins. MAGP1's ability to interact with plasma proteins was assayed by ligand blot (A) and coimmunoprecipitation (B). (A) Lanes: FN indicates fibronectin; VWF, von Willebrand factor; Fb, fibrinogen; and STD, molecular weight standards. The left side of the panel is a Coomassie blue–stained gel (± DTT) of the separated proteins. The right side shows a ligand blot of the same proteins after transfer to nitrocellulose, incubation with MAGP1, and bound MAGP1 detected with an antibody to MAGP1 after extensive washing to remove unbound protein. (B) SDS-PAGE autoradiogram showing coprecipitation of [¹²⁵I]-labeled plasma proteins and V5-tagged MAGP1. Lanes: 1, fibronectin with V5 antibody only (negative control); 2, fibronectin coprecipitated with MAGP1-V5 using V5 antibody; 3, fibronectin coprecipitated with MAGP1-V5 as in lane 2, but in the presence of 10-fold excess unlabeled fibronectin; 4, fibrinogen with V5 antibody; 5, fibrinogen coprecipitated with MAGP1-V5 using V5 antibody; 6, fibrinogen coprecipitated with MAGP1-V5 as in lane 5, but in the presence of 10-fold excess unlabeled fibrinogen; 7, von Willebrand factor with V5 antibody; 8, von Willebrand factor coprecipitated with MAGP1-V5 using V5 antibody; and 9, von Willebrand factor coprecipitated with MAGP1-V5 as in lane 8, but in the presence of 10-fold excess von Willebrand factor. Vertical lines have been inserted to indicate repositioned gel lanes.

Figure 7

Characterization of MAGP1 and VWF interactions using surface plasmon resonance. Different concentrations of von Willebrand factor were injected over MAGP1 immobilized on a BIAcore CM-5 sensor chip. Sensorgram shows 6 different analyte concentrations (0.052 μM, 0.105 μM, 0.21 μM, 0.32 μM, 0.42 μM, and 0.85 μM). One representative experiment is shown. The response difference (the difference between experimental and control flow cells) is given in resonance units (RU).