Analysis of platelet adhesion to a collagen-coated surface under flow conditions: the involvement of glycoprotein VI in the platelet adhesion

Abstract

Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials 7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) IIb/IIIa, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/IIIa activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.

Biological properties of a specific Gαq/11 inhibitor, YM‐254890, on platelet functions and thrombus formation under high‐shear stress

“…In thrombus formation on a collagen surface under high‐shear conditions, the initial interaction of vWF immobilized on a surface with GPIb/IX and subsequent vWF binding to GPIIb/IIIa is indispensable for proper mural thrombus growth (Moroi et al ., 1996; Tsuji et al ., 1999). In this study, YM‐254890 inhibited platelet thrombus formation on a collagen surface under high‐shear rate conditions in a flow chamber more potently than high‐SIPA in a cone‐and‐plate viscometer, suggesting the greater importance of G α q ‐mediated signal transduction for platelet thrombus growth on a collagen surface under high‐shear rate conditions.…”

Platelet glycoprotein VI‐related clinical defects

“…Glycoprotein VI‐related defects (Table I) are of the following types: (i) an acquired deficiency, resulting from (a) anti‐GPVI autoantibodies (Boylan et al , 2004; Sugiyama et al , 1987, 1993; Tsuji et al , 1997; Takahashi & Moroi, 2001) or (b) other causes (Nurden et al , 2004; Bellucci et al , 2005; Kojima et al , 2006); or (ii) a congenital deficiency, where (c) GPVI is not expressed (Moroi et al , 1989, 1996; Arai et al , 1995; Ryo et al , 1992; Tsuji et al , 1997) or (d) is expressed in a dysfunctional form with defective signalling to α IIb β 3 (Dunkley et al , 2007). While patient bleeding times might be normal or prolonged, the primary indication of GPVI‐related defects is a specific abnormality of platelet aggregation in response to collagen or other GPVI agonists (such as CRP or convulxin), with normal α IIb β 3 ‐dependent aggregation to other platelet agonists, such as ristocetin/von Willebrand factor (acting via platelet GPIb‐IX‐V) or ADP (acting via P2Y 1 and P2Y 12 ).…”

The GPIa C807T dimorphism associated with platelet collagen receptor density is not a risk factor for myocardial infarction

“…In whole blood, GPIa/IIa‐mediated platelet adhesion to fibrillar collagens activates platelets resulting in platelet aggregation mediated by GPIIb/IIIa and the generation of thrombin (Kirchhofer et al , 1995; Coller et al , 1989; Savage et al , 1990). Although the importance of GPIa/IIa in the early adhesive events is recognized, at least one other collagen receptor, possibly GPVI, is required for subsequent platelet activation (Moroi et al , 1996; Kehrel et al , 1998).…”