Contribution of platelet glycoprotein VI to the thrombogenic effect of collagens in fibrous atherosclerotic lesions - ScienceDirect

Atherosclerosis

Volume 181, Issue 1, July 2005, Pages 19-27

Atherosclerosis

https://doi.org/10.1016/j.atherosclerosis.2004.12.037 Get rights and content

Abstract

Collagens (types I and III) are among the strongest thrombus-forming components of the vascular subendothelium. We compared the thrombogenic effects of four collagen-containing advanced atherosclerotic lesions with those of purified types I and III collagen fibers. Cell-free homogenates from the human plaques effectively promoted platelet adhesion and aggregate formation under high-shear flow conditions, as well as exposure of procoagulant phosphatidylserine (PS) on platelets. With all plaques, blocking of the glycoprotein VI (GPVI) receptor for collagen abolished aggregation and PS exposure. Blocking of platelet ADP receptors resulted in similar, but less complete inhibitory effects. Type I collagen was more potent than type III collagen in inducing aggregation and PS exposure under flow, via stimulation of GPVI and ADP receptors. Type I collagen also more strongly enhanced thrombin generation with platelets and tissue factor, again via GPVI activation and PS exposure. The plaque material enhanced thrombin generation, partly due to the presence of tissue factor and partly via GPVI and ADP receptors. Together, these results indicate that in advanced plaques collagen type I is a major trigger of thrombus formation and PS exposure, acting via GPVI and ADP release, while tissue factor directly enhances coagulation.

Introduction

Fibrillar collagens, especially types I and III, are among the strongest platelet-adhesive and platelet-activating substances in the vascular subendothelium [1]. Two principal receptors are involved in platelet adhesion to collagen and subsequent activation, i.e. integrin α2β1 and the immunoglobulin superfamily member, glycoprotein VI (GPVI) [2]. The activating effect of direct platelet-collagen contact is extended by release of autocrine mediators from platelets, particularly ADP, which acts via the P2Y₁ and P2Y₁₂ receptors and leads to the formation of platelet aggregates [3], [4], [5]. Platelet interaction with collagen via GPVI also stimulates the procoagulant response, which is characterized by the expression of phosphatidylserine (PS) at the platelet outer membrane [6]. Exposed PS provides a catalytic surface for the assembly and activation of tenase and prothrombinase complexes and, thereby, enhances thrombin formation in plasma by several orders of magnitude [7].

Rupture or damage of an unstable atherosclerotic plaque leads to contact of the flowing blood with the material from the plaque core. Ensuing formation of platelet thrombi is considered to be a major cause of acute coronary syndrome and ischemic sudden death [8], [9]. In advanced plaque lesions, thrombogenic components are found particularly in the atheromatous core and the surrounding extracellular matrix [10], [11]. The core contains, in addition to cholesterol crystals, lipidic substances that activate platelets such as lysophosphatidic acid. The latter compound is also released by activated platelets themselves, and may act as a primary trigger in thrombus formation [12], [13]. Another thrombogenic substance on ruptured plaques is tissue factor (TF), which is a membrane-bound protein that, when de-encrypted, potently initiates the coagulation cascade [14], [15].

Atheromatous plaques are further rich in collagen fibers. Particularly the collagen types I, III and V increase during plaque development up to advanced fibrotic lesions [9], [16], [17]. Collagen in plaques has a structural function, as its degradation by metalloproteinases contributes to plaque instability [18]. However, collagen can also contribute to the thrombogenic reaction upon plaque rupturing. An early report describes that the increased thrombogenicity during plaque progression correlates with changes in quantity and nature of collagen types I and III [17]. However, until recently no suitable antibodies against platelet collagen receptors were available to evaluate the function of plaque collagens in thrombus formation. Precise information on the platelet-activating effect of plaque collagens is therefore still missing.

Recently, a monoclonal antibody (mAb), 9O12, against the collagen-binding domain of human GPVI has been developed. Fab fragments are highly effective in inhibiting collagen-induced secretion, aggregation and procoagulant activity of human platelets and, as a consequence, function as potent inhibitors of human thrombus formation [19], [20]. We anticipated that this antibody can also be used in determining the thrombogenic effect of plaque collagens. Here, we prepared cell-free material from a number of advanced human plaque lesions to investigate thrombus formation and platelet-dependent coagulation in the presence of this antibody. The thrombogenic effects of plaque materials were compared with those of purified types I and III collagens.

Section snippets

Materials

H-Phe-Pro-Arg chloromethyl ketone (PPACK) was obtained from Calbiochem (La Jolla, CA, USA). Bovine factor X, bovine serum albumin (BSA), MRS2179 (MRS), Trizma base and type III collagen were provided by Sigma (St. Louis, MO, USA). Z-Gly-Gly-Arg aminomethyl coumarin (Z-GGR-AMC) came from Bachem (Bubendorf, Switzerland), Oregon green-488 (OG488) labeled annexin A5 from Nexins Research (Hoeven, The Netherlands); recombinant human TF from Dade (Miami, FL, USA). The P2Y₁₂ receptor antagonist,

Characterization of used human plaques

Four human plaques, obtained at autopsy, were histochemically characterized as fibrous cap atheromas without signs of hemorrhage. Sections of these plaques were immunohistochemically stained for collagen types I and III, i.e. the major collagens in fibrous plaques and vessel wall [17], [27]. As shown for a typical example in Fig. 1, all plaques showed high staining for total collagen and type I collagen. Furthermore, the plaques stained positively for TF, using a blocking polyclonal antibody (

Discussion

The present results indicate that the examined homogenates from fibrous plaques are highly platelet-activating and procoagulant in nature. Collagen (type I) in plaques appears to be a potent trigger of thrombus formation, while TF in plaques enhances thrombin generation and coagulation. As collagen fibers and TF are found in the atheromatous plaque core, both thrombogenic substances may act in concert following plaque rupturing.

Flow experiments, carried out with anticoagulated whole blood,

Acknowledgements

We acknowledge A. Janssen and Dr. H. Spronk for histological analysis and stimulating discussions. We thank AgroBio (France) for supply of 9O12 antibody.

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