In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

Abstract

To examine the hypothesis that surface Pselectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin xllb183), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (Pselectin-positive, PKH2-labeled), 95 + 1% (mean + SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 ± 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKIH2-labeled, P-selectinnegative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.P-selectin, a member of the selectin family which includes Eand L-selectin, is a cell-adhesion molecule of activated platelets and endothelial cells (1, 2). P-selectin (also known as CD62P, GMP-140, and PADGEM protein) is a component of the a granule membrane of resting platelets that is only expressed on the platelet surface membrane during and after platelet degranulation and secretion (1,2). A soluble form of P-selectin circulates in plasma (3).Platelet surface P-selectin mediates the adherence of degranulated platelets to leukocytes in vitro (4, 5) and in vivo (6). It has therefore been postulated that surface P-selectin-positive (degranulated) platelets may be rapidly cleared from the circulation by leukocytes (2,4,5,7,8). In apparent contradiction to this postulate, other investigators have reported that degranulated platelets are no more rapidly cleared from the circulation than control platelets (9, 10). Methods have not previously been available to directly address this issue.In this study, we used novel three-color whole blood flow cytometric methods for tracking of circulating degran...

Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin

“…Furthermore, labeling of platelets with biotin at either level or with PKH26 or PKH67 did not affect the responsiveness of the platelets to ADP, collagen, or thrombin, even at the lowest concentrations of agonists that did not give maximal responses. Our findings with the PKH dyes using in vitro platelet function testing by aggregometry and measurement of dense granule release are in keeping with the observations of Michelson et al (10). They found that PKH2 labeling of baboon platelets did not alter function as measured by flow cytometry in in vivo and ex vivo studies.…”

“…For labeling with lipophilic fluorescent dyes, the method of Michelson et al (10) was followed, with some modifications introduced during preliminary experiments to prevent platelet aggregation. Platelets recovered by centrifugation of platelet-rich plasma were resuspended in Diluent C, with added 15 M PGI 2 , at a platelet count of 4 ϫ 10 9 /ml.…”

“…Some investigators have labeled platelets with PKH2 in vitro and identified them after infusion by flow cytometric analysis of their fluorescence (9,10). The lipophilic PKH dyes have long alkyl tails that intercalate into the lipid bilayer of cell membranes, as well as polar fluorescent head groups.…”

Quantification of ADP and ATP receptor expression in human platelets

“…It was recently demonstrated that activated platelets translate constitutive mRNAs into proteins [13][14][15]. Activation of platelets does not appear to decrease their life span [16][17][18], and this may be related to functional mRNAs in platelets. So it would be of interest to know the mRNA expression in platelets to better understand their gene regulatory mechanisms.…”

Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow

“…LS174T cells were labeled with 2 M red fluorescent lipophilic membrane stain PKH26 according to the manufacturer's (Sigma) protocol, and platelets were similarly stained with 2 M green fluorescent PKH67 (Sigma) (28). HUVECs were incubated with 10 g/ml anti-ICAM-1 MAb for 30 min at 37°C before use in flow chamber assays.…”