Collagen But Not Fibrinogen Surfaces Induce Bleb Formation, Exposure of Phosphatidylserine, and Procoagulant Activity of Adherent Platelets: Evidence for Regulation by Protein Tyrosine Kinase-Dependent Ca2+ Responses

Abstract

With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

Adhesion of human and mouse platelets to collagen under shear: a unifying model

“…The effect of PD0173952 on a number of additional consequences of platelet interaction with collagen was assessed. One of the earliest GPVI-induced platelet responses is an elevation of [Ca 2+ ] i (30). For both human and mouse Fluo-3 loaded platelets, averaged increases in [Ca 2+ ] i following adhesion to collagen under shear were completely inhibited in the presence of 25 µM PD0173952 ( Fig.…”

“…Microscopic bright-field phase-contrast images and nonconfocal fluorescence images were recorded from flow chambers with a two camera system, as described previously (30). Where indicated, Fluo-3-loaded platelets were added to the blood, and a series of images was recorded at 5 Hz with the same system for 2 min.…”

Complementary roles of platelet glycoprotein VI and integrin α2β1 in collagen‐induced thrombus formation in flowing whole blood ex vivo

“…During the perfusion, high-resolution microscopic transmission and/or fluorescent fluo-3 images were recorded in real time via a Visitech imaging system (Sunderland, UK). Digital images were captured with two parallelplaced intensified, charge-coupled device cameras, recording phase-contrast light and epifluorescence (16). After 4 min, the flow chamber was perfused with HEPES buffer supplemented with 1 U/ml heparin at the same shear rate for 10 min.…”

“…The importance of the FcR γ chain is exemplified by the fact that mice lacking this protein fail to express GPVI on their platelets and do not respond to collagen (14,15). Signaling through the FcR γ chain activates phospholipase Cγ2 via tyrosine kinases, which causes a prolonged increase in cytosolic [Ca 2+ ] i (7,16). This Ca 2+ response not only mediates exposure of procoagulant PS but also converts the platelets into blebbing cells, from which procoagulant microvesicles are shed (16,17).…”

“…Signaling through the FcR γ chain activates phospholipase Cγ2 via tyrosine kinases, which causes a prolonged increase in cytosolic [Ca 2+ ] i (7,16). This Ca 2+ response not only mediates exposure of procoagulant PS but also converts the platelets into blebbing cells, from which procoagulant microvesicles are shed (16,17).…”