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.

Accumulation of monocyte-derived foam cells in focal areas of the atherosclerotic (A.S.-) lesion is one of the key events in early atherogenesis. Using a flow model for the damaged vessel wall, we examined the ability of ECM-bound platelets to induce monocyte tethering and adhesion. Whereas ECM-proteins alone induced monocyte adhesion only at low shear stresses (F100 mPa), ECM-bound platelets induced monocyte rolling and adhesion at shear stresses up to 240 mPa. Studies with specific antibodies showed that monocyte adhesion to platelets was mainly mediated by P-selectin and monocyte PSGL-1 (maximum inhibition 90%). ␤ 2 -Integrin blocking CD18 and CD11b antibodies partly inhibited the arrest of rolling cells. Antibodies against other adhesion molecules such as LFA-1, PECAM-1, and ␤ 1 -integrins had no effect. Even sparsely adhered platelets (D10% coverage of the surface) already strongly supported monocyte tethering. In conclusion, activated platelets present on ECM are a powerful adhesive substrate for monocyte recruitment under flow conditions. J. Leukoc. Biol. 64: 467-473; 1998.

SummarySurface-bound platelets support selectin-mediated rolling and β2-integrin-mediated firm adhesion of neutrophils (PMN) under flow conditions. We examined which ligands on platelets mediate this firm adhesion. Surface-bound platelets express ICAM-2 and GPIIbIIIa-bound fibrinogen, which are ligands for LFA-1 and MAC-1. In a well defined model for vessel wall injury, blood from an afibrinogenemic patient was perfused over ECM-coated coverslips to obtain fibrinogen-free platelet surfaces. At high shear rates, PMN-adhesion to fibrinogen-free platelet surfaces decreased compared to fibrinogen-containing controls. Under these conditions, firm adhesion and not rolling was blocked demonstrating the importance of fibrinogen in this process. In addition, MAC-1 and LFA-1 on PMN and ICAM-2 on platelets played a role in firm adhesion; the effect of blocking antibodies was most evident at high shear. The effects of fibrinogen depletion and ICAM-2 blocking were additive. In conclusion, multiple redundant ligands, like ICAM-2 and fibrinogen, induce firm and shear resistant PMN adhesion to platelets under flow conditions. Individually these ligands become critical at higher shear. Blocking of two or more interactions also interferes with low shear adhesion.

At sites of vessel wall damage, the primary hemostatic reac-(320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN tion involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Ad-adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone hered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)-threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion.However, at higher shear rates (Û200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interac-Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling tion of PMNs with a fibrin network.

The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by β2-integrins. However, the influence of physiologic flow conditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil activation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of ) neutrophil adhesion, real-time video-assisted microscopy and custom-made software were used. Under flow conditions, adherent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the adherence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10−7 mol/L) or 4β-phorbol, 12-myristate, 13-acetate (PMA; 100 ng/mL) resulted in approximately 50% inhibition of adhesion to fibrin and a more random distribution (cluster index <0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respectively. Under these conditions, anti–L-selectin antibodies or neuraminidase did not affect adhesion. These results indicate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin–mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interaction. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation.

Abstract-Platelets at injured vessel wall form an adhesive surface for leukocyte adhesion. The precise relation between platelet adhesion and activation and leukocyte adhesion, however, is not known. We therefore used various models of injured vessel wall to form different patterns of platelet adhesion. The interaction of polymorphonuclear neutrophils (PMNs) was subsequently studied under flow conditions. In the absence of platelets, not only endothelial cell, smooth muscle cell, and fibroblast matrices but also purified matrix proteins (fibrinogen, collagen, and fibronectin) barely support PMN adhesion. The presence of platelets, however, strongly enhances PMN adhesion. PMN adhesion shows a proportional increase with platelet coverage up to 15%. Although PMNs roll over the scarcely scattered platelets, they speed up again when encountering surfaces without platelets. This "hopping" interaction of PMNs vanishes with platelet coverage Ͼ15%. Unobstructed rolling of PMNs is than observed and soon leads to a maximal adhesion of 1000 to 1200 cells/mm 2 . The mean rolling velocity of PMNs continues to decrease with higher platelet coverage. Platelet aggregate formation is an accepted indicator of platelet activation. The presence of platelet aggregates instead of contact or spread platelets, however, does not increase PMN adhesion. Also, additional stimulation of surface-associated platelets by thrombin fails to influence PMN adhesion. Moreover, indomethacin as an inhibitor of platelet activation and aggregation does not change the subsequent PMN interaction. In conclusion, approximately 15% of platelet coverage is sufficient for optimal PMN adhesion. Increasing platelet coverage increases the availability of platelet-associated receptors that lower PMN rolling velocity. Additional activation of adherent platelets makes no difference in the expression of relevant adhesion receptors. Therefore, minimal vascular damage in vivo and only scarce platelet adhesion will already evoke significant colocalization of leukocytes. Key Words: platelet adhesiveness Ⅲ thrombosis pathophysiology Ⅲ vascular injury Ⅲ leukocyte adhesion Ⅲ flow P latelet adhesion at the injured vessel wall was convincingly shown to support leukocyte adhesion under flow conditions. 1-3 The observed colocalization of the hemostatic and inflammatory response in thrombosis, vasculitis, and atherosclerosis in vivo supports the role of these interactions in pathophysiology of these diseases. 4 -6 Recent research both in vivo and in vitro in perfusion chambers showed that platelets at the injured vessel wall form an extremely powerful substrate for leukocyte, eg, polymorphonuclear neutrophil (PMN) adhesion under flow conditions. The first step in PMN adhesion consists of a rolling or tethering interaction involving P-selectin 1-3 expressed by activated platelets and P-selectin glycoprotein ligand-1 by leukocytes. Firm adhesion follows by integrin-ligand interactions. Mac-1 (CD11bCD18) was identified to be the main leukocyte ␤2 integrin that binds to the platele...

At sites of vessel wall damage, the primary hemostatic reaction involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Adhered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)-bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (<200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interaction of PMNs with a fibrin network.

The murine chronic proliferative dermatitis mutation (cpdm/cpdm) is characterized by epidermal hyperplasia and hyperproliferation of ventral and dorsal skin sites. The expression of endothelium-associated adhesion molecules was studied in combination with the binding capacity of various cell types on frozen sections of the affected skin. In correlation with the relative absence of lymphocytes in the cpdm/cpdm skin no lymphocyte binding could be observed, but avid adhesion of neutrophils was seen. Binding of neutrophils could be blocked with antibodies against L-selectin, LFA-1, CR3 and anti ICAM-1. No expression of vascular addressins or E-selectin on endothelium in the dermis was found. The cpdm/cpdm mutation has therefore characteristics of a psoriasis-like as well as a more generalized inflammatory skin condition.