Abstract-We examined the effect of adherent neutrophils on fibrin deposition under laminar flow conditions. Perfusion of recalcified citrated platelet-free plasma (PFP) over neutrophils adherent to fibrinogen-coated glass at a venous wall shear rate of 62.5 s Ϫ1 for 15 minutes resulted in dense deposition of fibrin around each neutrophil, whereas fibrin deposition on glass alone was sparse. Fibrin deposition on neutrophils was markedly reduced by anti-CD18 or anti-CD11b or a higher shear rate (250 s Ϫ1 ). Significantly less fibrin was deposited around adherent fibrinogen-coated beads, indicating that nonspecific "cross-sectional capture" effects were not responsible for the massive fibrin deposition on neutrophils. Direct visualization of fibrin capture by neutrophils and elimination of fibrin deposition at 15 minutes by a factor XIIa inhibitor (50 g/mL corn trypsin inhibitor [CTI]) or elastase/cathepsin G inhibitors (MethoxysuccinylAla-Ala-Pro-Ala-Chloromethyl-Ketone/Z-Gly-Leu-Phe-CMK, 100 mol/L) indicated that neutrophils can capture short fibrin strands flowing in recalcified PFP lacking CTI and can also promote thrombin generation through pathways attenuated by inhibitors of factor XIIa, elastase, and cathepsin G. When neutrophils were allowed to interact with platelets on a fibrinogen surface before perfusion of recalcified CTI-treated PFP, the fibrin deposition was observed to be dramatic compared with that over surfaces coated with platelets alone or neutrophils alone and compared with that formed on platelets adherent to collagen. This neutrophil promotion of platelet-mediated fibrin formation was attenuated by inhibitors of elastase or cathepsin G but not anti-tissue factor antibody. Neutrophils can interact with platelets via released proteases to increase platelet procoagulant activity and fibrin formation in CTI-treated plasma under the low-flow conditions expected in venous thrombosis or inflammation. Key Words: P-selectin Ⅲ CD11b/CD18 Ⅲ thrombin Ⅲ deep vein thrombosis B lood coagulation on the inner wall of blood vessels is the major cause of cardiovascular diseases, such as deep vein thrombosis, myocardial infarction, and stroke. In a Dacron graft model, Palabrica et al 1 showed that neutrophils enhance fibrin deposition through P-selectin-dependent mechanisms. Adherent neutrophils may promote thrombosis via pathways such as deencryption of tissue factor, 2 release of proteases that activate platelets, 3,4 Mac-1 (CD11b/CD18) binding of factor X, 5 and improved capture of flowing platelets via protrusion into the flow stream. 6 The capture of neutrophils by the vessel wall involves neutrophil P-selectin glycoprotein ligand-1 (PSGL-1)-mediated rolling 7 and membrane tethering 8 on P-selectin presented by activated endothelium or spread platelets, 9 followed by  2 -integrin-mediated firm arrest. 10 The neutrophil  2 -integrin Mac-1, when upregulated, can bind endothelial intercellular adhesion molecule-1 and an unknown platelet ligand, 11 which may be glycoprotein Ib␣. 12 Adherent neutrophils can a...
Plasminogen activators (PAs; e.g., tissue-type, tPA) coupled to red blood cells (RBCs) display in vivo features useful for thromboprophylaxis: prolonged circulation, minimal extravasation, and preferential lysis of nascent versus preexisting clots. Yet, factors controlling the activity of RBC-bound PAs in vivo are not defined and may not mirror the profile of soluble PAs. We tested the role of RBC/PA binding to fibrin in fibrinolysis. RBC/ tPA and RBC/tPA variant with low fibrin affinity (rPA) bound to and lysed plasminogen-containing fibrin clots in vitro comparably. In contrast, when coinjected in mice with fibrin emboli lodging in pulmonary vasculature, only RBC/tPA accumulated in lungs, which resulted in a more extensive fibrinolysis versus RBC/rPA (p Ͻ 0.01). Reconciling this apparent divergence between in vitro and in vivo behaviors, RBC/tPA, but not RBC/rPA perfused over fibrin in vitro at physiological shear stress bound to fibrin clots and caused greater fibrinolysis versus RBC/rPA (p Ͻ 0.001). These results indicate that because of high fibrin affinity, RBC/tPA binding to clots endures hemodynamic stress, which enhances fibrinolysis. Behavior of RBC/PAs under hemodynamic pressure is an important predictor of their performance in vivo.
Human neutrophil proteases cathepsin G and elastase can directly alter platelet function and/or participate in coagulation cascade reactions on the platelet or neutrophil surface to enhance fibrin formation. The clotting of recalcified platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (to shut down contact activation) was studied in wellplates or flow assays. Inhibitors of cathepsin G or elastase significantly delayed the burst time (t 50 ) of thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets under flow conditions. In flow assays, purified cathepsin G was a far more potent activator of platelet-dependent coagulation than elastase. Anti-tissue factor had no effect on neutrophil protease-enhanced thrombin formation in PRP. The addition of cathepsin G (425 nM) or convulxin (10 nM) to PRP dramatically reduced the t 50 of thrombin generation from 53 min to 17 or 23 min, respectively. In contrast, the addition of elastase to PRP left the t 50 unaltered. Whereas perfusion of PFP (␥ w ؍ 62.5 s ؊1 ) over fibrinogen-adherent platelets did not result in fibrin formation until 50 min, massive fibrin could be observed on cathepsin G-treated platelets even at 35 min. Cathepsin G addition to corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of fibrinogen-adherent platelets rather than by cleaving coagulation factors.
Summary. Background: Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets. Objectives: The objective of this study was to examine if platelets, following activation of GPVI, could support tenase and prothrombinase activity without any exogenously added tissue factor. Methods: Thrombin (IIa) formation on gel‐filtered platelets with added factors or the clotting of platelet‐free plasma (PFP) or platelet‐rich plasma (PRP) supplemented with corn trypsin inhibitor (CTI) (to inhibit factor XIIa) was studied in well plate assays with a fluorogenic thrombin substrate or in flow assays by fibrin visualization. Results: Pretreatment of convulxin (CVX)‐stimulated, fibrinogen‐adherent, gel‐filtered platelets with anti‐TF, anti‐FVII/VIIa, or 1 nm PPACK [inhibitor of FVIIa, factor XIa and factor (F)IIa] delayed fibrin deposition on platelets perfused with PFP/CTI at 62.5 s−1. Anti‐TF or anti‐FVII/VIIa also attenuated thrombin generation in plate assays using recalcified PRP/CTI treated with CVX. Anti‐TF or anti‐FVII/VIIa (but not inhibited factor IXa) delayed the burst in thrombin production by gel‐filtered platelets suspended in prothrombin and CVX by 14 min and 40 min, respectively. Anti‐FVII/VIIa completely eliminated thrombin generation on fibrinogen‐adherent, gel‐filtered platelets pretreated with 10 µm PPACK and 10 µm EGR‐CK [inhibitor of factor (F)Xa], rinsed, and then supplemented with CVX, prothrombin, and FX. Addition of anionic phospholipid to PFP/CTI or to a mixture of prothrombin, FX, and recVIIa was not sufficient to generate detectable tenase activity. Lastly, isolated, unactivated neutrophils suspended in FX, FII and recVIIa supported a very low level of thrombin generation sensitive to antagonism of P‐selectin, CD18, and TF. Conclusions: Activated platelets supported tenase and prothrombinase activity by elevating the function or level of FVIIa and exposing active FVIIa or FVIIa‐cofactor(s), distinct from anionic lipid, that may be, in part, TF.
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