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Platelet-Dependent Primary Hemostasis Promotes Selectin- and Integrin-Mediated Neutrophil Adhesion to Damaged Endothelium Under Flow Conditions

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Platelet-Dependent Primary Hemostasis Promotes Selectin- and Integrin-Mediated Neutrophil Adhesion to Damaged Endothelium Under Flow Conditions Platelet-Dependent Primary Hemostasis Promotes Selectin- and Integrin-Mediated Neutrophil Adhesion to Damaged Endothelium Under Flow Conditions By P.H.M. Kuijper, H.I. Gallardo Torres, J.A.M. van der Linden, J.-W.J. Lammers, J.J. Sixma, L. Koenderman, and J.J. Zwaginga Co-localization of blood platelets and granulocytes at sites PMN adhesion to unstimulated HUVEC was only substantial of hemostasis and inflammation has triggered an intense at low shear (up to 200 cells/mm2 at shear stress BO mPa). interest in possible interactions between these cellular pro- In marked contrast, PMN adhesion to ECM-adhered platelets cesses and induction of vessel wall injury. Leukocyte adhe- was dramatically increased, and adhesion was demon- sion to endothelial cells decreases with increasing shear and strated at much higher shear stress (up to 640 mPa1. Studies is dependent on an initial rolling phase mediated by selec- with specific antibodies showed that the platelet-dependent tins. We hypothesized that flow-dependent platelet adhe- neutrophil adhesion was selectin-mediated. inhibition of P- sion at an injured vessel wall will lead to P-selectin expres- selectin caused a marked inhibition of adhesion at high shear sion by platelets, thus mediating leukocyte co-localization. stress, whereas the role of leukocyte L-selectin was less pro- A perfusion chamber was used in which flowing whole blood nounced. P,-lntegrin-blocking antibodies inhibited static induced platelet adhesion to a subendothelial matrix (ECM) neutrophil adhesion. fMLP induced L-selectin shedding from Downloaded from http://ashpublications.org/blood/article-pdf/87/8/3271/620932/3271.pdf by guest on 29 September 2021 of cultured human umbilical vein endothelial cells (HUVEC). leukocytes, resulting in decreased leukocyte adhesion. In We compared neutrophil (polymorphonuclear leukocyte conclusion, platelet-dependent hemostasis at the ECM ap- [PMN]) interactions with HUVEC and their ECM with and pears to be a powerful intermediate in neutrophil-vessel without adhered platelets. PMNs adhered predominantly to wall interactions at shear stresses that normally do not ECM-adhered platelets and not to endothelial cells. ECM allow neutrophil adhesion to intact endothelium. alone did not support PMN adhesion under flow conditions. 0 1996 by The American Society of Hematology. HE PHYSIOLOGY OF hemostasis and inflammation, rapid platelet adhesion, activation, and immediate expression T as well as the pathophysiology of thrombosis, vasculi- of P-se1e~tin.l~In vivo observations in atherosclerosis, vas- tis, and metastatic seeding, involves adhesion of cells sus- culitis, acute cardiovascular accidents, and thrombosis have pended in flowing blood to the vessel wall. Extensive re- shown that both local platelet adhesion and large numbers search has shown the importance of flow and of exposed of leukocytes are found at these sites.''." Moreover, it has vessel wall structures in these processes. In this respect, been shown that activated platelets in suspension or bound to platelet-dependent hemostasis at injured vessel walls is more artificial structures support leukocyte adhesion in a selectin- extensive at higher shear. However, adhesion of leukocytes dependent In line with these reports, we hypothe- to endothelium decreases at higher shear.' Multiple receptor- sized that exposure of a subendothelial surface to blood leads ligand interactions are usually needed for firm attachment.' to acute platelet adhesion and P-selectin expression. This Membrane-associated selectins mediate the initial margina- platelet-covered vessel wall surface may then allow leuko- tion and rolling, but activated &integrins (CD18 com- cyte adhesion that can be highly shear-resistant. plexes) are necessary for subsequent firm attachment (or In this study, a perfusion system was used in which flow- static adhesion) and ~preading.~P- and E-selectin are ex- ing whole blood was exposed to endothelium or ECM. In pressed on endothelial cells upon stimulation, and activated the latter, the mimicking of physiologic conditions leads to platelets express P-selectin as well. Leukocytes constitu- a homogeneous platelet adhesion. We subsequently exam- tively express L-selectin, which can be quickly shed from ined the interaction of isolated neutrophils (polymorphonu- the membrane upon activation. Selectins recognize oligosac- clear leukocytes [PMNs]) under flow conditions with (dam- charide-based ligands such as sialyl-Lewis X in a calcium- aged) vessel wall components, such as endothelial cells, dependent This interaction is sensitive to inhibition ECM, and ECM-adhered platelets. The influence of shear by heparin oligosaccharide^.^^^ The influence of P,-integrins on the rolling and adhesion of isolated PMNs to these sur- is clear from the deficient inflammation-induced extravasa- tion of neutrophils seen in leukocyte adhesion deficient From the Departments of Haematology and Pulmonaty Diseases, (LAD) patients8 Transgenic mice, deficient in P- and L- University Hospital Utrecht, Utrecht, The Netherlands. selectin, illustrate the importance of selectins for leukocyte Submitted June 19, 1995; accepted December 6, 1995. extravasation in vivo.' Supported by Dutch Heart Foundation Grant No. 92.106 and So far, in vitro experiments suggest that high or arterial by a research grant from Glaxo Wellcome BV, The Netherlands shear virtually eliminates binding of granulocytes to endo- (J.A.M.v.d.L.). thelium-even after appropriate stimulation." However, ob- Address reprint requests to J.J. Zwaginga, MD, Department of servations in vivo show that vascular inflammation is cer- Huematology, Room G.03.647, University Hospital Utrecht, Heidel- tainly not limited to vessels in which a low shear is found." berglaan 100, 3584 CX Utrecht, The Netherlands. Together with the presence of platelet deposition at high The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked shear inflammatory these observations prompted us "advertisement" in accordance with 18 U.S.C. section 1734 solely to to study the role of platelets in leukocyte adhesion to a indicate this fact. damaged vessel wall under high-shear conditions. Here, ex- 0 1996 by The American Society of Hematology. tracellular matrix (ECM) exposure to flowing blood leads to OO06-4971/96/8708-00I3$3. 00/0 Blood, Vol 87, No 8 (April 15). 1996: pp 3271-3281 327 1 3272 KUIJPER ET AL faces was evaluated with real-time video-assisted image stress compared with cells grown on gelatin-coated cover slips, as analysis. We show that ECM-adhered platelets are a better described by Reutelingsperger et al.*’ HUVEC confluency and mor- adhesive substrate for neutrophils, compared with cultured phology were checked by phase-contrast microscopy before and after endothelial cells. We hypothesize that in vivo, the primary experiments. Confluent HUVEC were used in perfusion experiments hemostatic reaction involving platelet deposition provides with whole blood and neutrophils. In some cases, HUVEC were stimulated with tumor necrosis factor alpha (TNF-a) before the ex- intriguing mechanism for leukocyte recruitment at the an periment. Therefore TNF-a (100 UhL) was diluted in fresh culture damaged vessel wall. medium and incubated for 6 hours at 37°C. Surface ECM coating. Cover slips with ECM were obtained as MATERIALS AND METHODS described previously.” In short, HUVEC were cultured on 1% gela- Monoclonal antibodies. The monoclonal antibody (MoAb) tin-coated cover slips. Cell monolayers were grown to confluence CLB/Thromb-6 (anti-P-selectin, CD62p)I5 was kindly provided by in 5 to 7 days. The matrix was obtained after removal of endothelial Dr A. Von dem Borne (Central Laboratory of The Netherlands Red cells by exposure to 0.1 mom NH,OH for 5 minutes at room temper- Cross Blood Transfusion Service, Amsterdam, The Netherlands). ature. Isolated matrices were washed and kept in phosphate-buffered The MoAb LAMl-3 (anti-L-selectin, CD621, 5 pg/mL for blocking saline ([PBS] 1:10 voUvol 0.1-mol& sodium phosphate buffer in studies) was a kind gift from T. Tedder (Harvard Medical School, 150 mmom NaC1) at 4°C for a maximum of 3 weeks. Boston, MA). The MoAb WASP122 (anti-P-selectin, CD62p, 1 Surface platelet-covered HlJVEC/ECM. Platelet adherence to Downloaded from http://ashpublications.org/blood/article-pdf/87/8/3271/620932/3271.pdf by guest on 29 September 2021 pg/mL for blocking studies) was purchased from Endogen (Boston, different surfaces was accomplished by perfusing whole blood from MA). MoAbs 44a (CDI lb, 10 pg/mL for blocking studies) and IB4 healthy volunteer donors (Blood Bank, Utrecht, The Netherlands) (CD18, 10 pg/mL for blocking studies) were purchased from the as described previo~sly.’~The blood was anticoagulated with ‘/,,,th American Type Culture Collection (Rockville, MD). The MoAb vol 1 10 mmol/L trisodium citrate. When a monolayer of HUVEC Leu4 (anti-L-selectin, CD621) and CSLEXl (anti-sialylLe”) were were used as a surface, whole blood was perfused at 37°C for 5 from Becton Dickinson (San Jose, CA). The MoAb 80H3 (CD66b) minutes at a shear rate of 400 s-’, which corresponds with a shear was purchased from Immunotech (Marseille, France). stress of approximately 2 Pa with a blood viscosity of 5 mPa. s. At Reagents. Percoll was obtained from Pharmacia (Uppsala, Swe- this shear rate, the HUVEC monolayer stayed largely intact, while den). MLP, thrombin, and 4P-phorbo1, 12-myristate, 13-acetate at higher shear rates HUVEC are progressively shedded from the (PMA) were purchased from Sigma (St Louis, MO). Experiments matrix. To induce platelet adhesion to ECM-coated cover slips, 15 were performed in HEPES buffer (20 mmol/L HEPES, 132 mmol/ mL whole blood was perfused at 37°C for 7 minutes at a shear rate L NaCI, 6 mmol/L KCI, 1.2 mmol/L KH,PO,, 1 mmol/L of 1,600 s ’ (ie, a shear stress of -7 Pa) in a recirculating system. MgSO,. 7H20, 5 mmol/L glucose, and 1 mmol/L CaCl,, pH 7.4). After completion of the whole-blood perfusion, cover slips were HEPES buffers with different free-calcium concentrations were rinsed with HEPES buffer to remove blood remnants.
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