Platelet-dependent primary hemostasis promotes selectin- and integrin- mediated neutrophil adhesion to damaged endothelium under flow conditions

Abstract

Co-localization of blood platelets and granulocytes at sites of hemostasis and inflammation has triggered an intense interest in possible interactions between these cellular processes and induction of vessel wall injury. Leukocyte adhesion to endothelial cells decreases with increasing shear and is dependent on an initial rolling phase mediated by selectins. We hypothesized that flow-dependent platelet adhesion at an injured vessel wall will lead to P-selectin expression by platelets, thus mediating leukocyte co-localization. A perfusion chamber was used in which flowing whole blood induced platelet adhesion to a subendothelial matrix (ECM) of cultured human umbilical vein endothelial cells (HUVEC). We compared neutrophil (polymorphonuclear leukocyte [PMN]) interactions with HUVEC and their ECM with and without adhered platelets. PMNs adhered predominantly to ECM-adhered platelets and not to endothelial cells. ECM alone did not support PMN adhesion under flow conditions. PMN adhesion to unstimulated HUVEC was only substantial at low shear (up to 200 cells/mm2 at shear stress 80 mPa). In marked contrast, PMN adhesion to ECM-adhered platelets was dramatically increased, and adhesion was demonstrated at much higher shear stress (up to 640 mPa). Studies with specific antibodies showed that the platelet-dependent neutrophil adhesion was selectin-mediated. Inhibition of P-selectin caused a marked inhibition of adhesion at high shear stress, whereas the role of leukocyte L-selectin was less pronounced. beta2-Integrin-blocking antibodies inhibited static neutrophil adhesion. fMLP induced L-selectin shedding from leukocytes, resulting in decreased leukocyte adhesion. In conclusion, platelet- dependent hemostasis at the ECM appears to be a powerful intermediate in neutrophil-vessel wall interactions at shear stresses that normally do not allow neutrophil adhesion to intact endothelium.

Circulating platelet–neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing

“…This may be explained by considering the role of neutrophil integrins in complex formation. A number of groups have observed the capacity of immobilized platelets to bind leucocytes (including neutrophils) via a multistep selectin and b2 integrin dependent mechanism (Kuijper et al, 1996(Kuijper et al, , 1997Buttrum et al, 1993;Diacovo et al, 1996), analogous to that which occurs between activated endothelium and leucocytes. Platelet CD62P, platelet-derived PAF and neutrophil b2-integrin activation have recently been described as requirements for maximal adhesion of neutrophils on immobilized platelets (Evangelista et al, 1996).…”

Ischemia–reperfusion injury pathophysiology, part I

“…These platelets may then recruit additional neutrophils by initiating selectin-and integrin-dependent leukocyte adhesion to surface-bound platelets. [113][114][115] The accumulated neutrophils adhering to the platelets may promote fibrin deposition and subsequent thrombus formation. 116 Since there is often a period of normal flow after elimination of the occlusion and a subsequent decrease in flow, the term diminishing reflow has been suggested.…”

P-selectin and MAC-1 mediate monocyte rolling and adhesion to ECM-bound platelets under flow conditions

“…Monocyte perfusions were performed and evaluated as described previously for polymorphonuclear leukocytes (PMNs) [22]. In short, during the perfusion the flow chamber was mounted on a microscope stage (DM RXE, Leica, Weitzlar, Germany) that was equipped with a B/W CCD-video-camera (Sanyo, Osaka, Japan) coupled to a VHS video recorder.…”

“…The number of surface-adhered monocytes was measured after 5 min of perfusion at a minimum of 30 randomized high-power fields (total surface Ͼ 3 mm 2 ). The rolling velocity of cells was measured as described before for PMNs [22]. In short, a sequence of images was digitally captured with a pre-set time interval (␦t).…”

“…The surfaces covered with ECM-bound platelets were obtained by perfusion of whole blood over ECM-coated coverslips at a high shear stress (4 Pa ϭ 40 dynes/cm 2 ; shear rate is 1600 s -1 ) for 7 min. Under given conditions, homogeneous platelet coverage of more than 85% of the surface was accomplished [22]. Monocyte adhesion to ECM surfaces with or without bound platelets was examined at different shear stresses ( Fig.…”