Objective-Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. Methods and Results-If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7Ϯ1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) Key Words: blood cells Ⅲ fibrin Ⅲ fibrinolysis Ⅲ plasminogen activators Ⅲ platelet receptor blockers A reverse correlation between bleeding time and red blood cell (RBC) count was observed more than a century ago. 1 When anemic and thrombocytopenic patients were treated with transfusion, the correction of the bleeding time correlated with the corrected hematocrit despite the fall in the platelet count to pretransfusion levels. Later studies 2 confirmed that the prolonged bleeding time of severely anemic patients can be corrected with washed RBC transfusion (thus excluding the effect of plasma factors) at essentially unchanged platelet counts. The mechanism of RBC contribution to hemostatic function is still open to question, notwithstanding their known role in the maintenance of blood viscosity, the chemical signaling of platelet activation, and the provision of phospholipid support for the activation of coagulation factors (reviewed by Wohner 3 ). The solid matrix of hemostatic and pathological blood clots is composed of fibrin and variable cellular elements, but the incorporation of RBCs appears to be mediated through a less specific entrapment than that of platelets. 4 Based on binding data, approximately 2% of fibrinogen, the plasma precursor of fibrin monomers, is estimated to circulate in vivo in association with erythrocytes. 5 This interaction is not simply a nonspecific protein adhesion to the cell membrane, but it involves an erythrocyte receptor 6 and a specific domain around residues 207 to 303 of fibrinogen A␣-chains. 7 Recently, the RBC receptor for fibrinogen has been identified as an integrin related to the platelet ␣ IIb ␤ 3 receptor with similar eptifibatide sensitivity and impaired function in Glanzmann thrombasthenia. 8 When fibrinogen is converted to fibrin, the presence of RBCs modifies the structural and viscoelastic characteristics of plasma clots. 9,10 Because fibrin structure profoundly affects the subsequent removal of blood clots from the vasculature by tissue plasminogen activator (tPA)-dependent proteolysis as a basic fibrinolytic mechanism (reviewed recently by Weisel and Litvinov 11 and Lord 12 ), the presence of RBCs may change the lytic susceptibility of thro...

Summary Quantitative deficiencies in von Willebrand factor (VWF) are associated with abnormal hemostasis that can manifest in bleeding or thrombotic complications. Consequently, many studies have endeavored to elucidate the mechanisms underlying the regulation of VWF plasma levels. This review focuses on the role of VWF clearance pathways. A summary of recent developments are provided, including results from genetic studies, the relationship between glycosylation and VWF clearance, the contribution of increased VWF clearance to the pathogenesis of von Willebrand disease and the identification of VWF clearance receptors. These different studies converge in their conclusion that VWF clearance is a complex phenomenon that involves multiple mechanisms. Deciphering how such different mechanisms coordinate their role in this process is but one of the remaining challenges. Nevertheless, a better insight into the complex clearance pathways of VWF may help us to better understand the clinical implications of aberrant clearance in the pathogenesis of von Willebrand disease and perhaps other disorders as well as aid in developing alternative therapeutic approaches.

Although fibrin forms the core matrix of thrombi, their structure depends also on the cellular elements embedded in its meshwork. Platelets are essential in the initial stages of thrombus formation, because they adhere and aggregate at sites of blood vessel wall injury and then serve as a surface for coagulation reactions, the overall rate of which determines the final structure of fibrin. In addition, platelets affect fibrinolysis through their proteins and phospholipids, which modulate plasmin activity. Leukocytes form mixed aggregates with platelets and thus influence the structure of thrombi. After activation they secrete different proteases (elastase, cathepsin G, matrix metalloproteinases) that enhance the von Willebrand factor-dependent platelet adhesion. Leukocyte-derived enzymes, first of all elastase, effect fibrinolysis by direct digestion of fibrin or indirectly modulate it by partial degradation of zymogens and inhibitors of coagulation and fibrinolytic proteases.

Summary. Background: von Willebrand factor (VWF) is cleared in a shear stress-and macrophage-dependent manner by LRP1. von Willebrand disease (VWD)-type 2B mutants are endocytosed more efficiently than wildtype (wt)-VWF by macrophages. Objective: To investigate if VWD-type 2B mutations in the VWF A1-domain affect LRP1 binding and LRP1-dependent clearance. Methods: Recombinant Fc-tagged A1 domain (A1-Fc, A2-Fc, A3-Fc) and full-length VWF (wt or mutants thereof) were tested for binding to LRP1 or a recombinant fragment thereof in a static immunosorbent assay. Mutant and wt-VWF were also compared for clearance in mice lacking macrophage LRP1 (macLRP1 À ) and control mice (macLRP1 + ). Results: We found that A1-Fc but not A2-Fc or A3-Fc binds dose-dependently to LRP1. Binding of A1-Fc to LRP1 was markedly enhanced by the VWD-type 2B mutation p.V1316M. As expected, full-length wt-VWF was unable to bind LRP1 under static conditions unless ristocetin was added. In contrast, the presence of the p.V1316M or p.R1306Q mutation induced spontaneous binding to LRP1 without the need for ristocetin or shear stress. Both mutants were cleared more rapidly than wt-VWF in control macLRP1 + mice. Surprisingly, deletion of macrophage LRP1 abrogated the increased clearance of the VWF/p.R1306Q and VWF/p.V1316M mutant. Conclusion: The VWF A1-domain contains a binding site for LRP1. Certain VWD-type 2B mutations relieve the need for shear stress to induce LRP1 binding. Enhanced LRP1 binding coincides with a reduced survival of VWF/p.R1306Q and VWF/p.V1316M. Our data provide a rationale for reduced VWF levels in at least some VWD-type 2B patients.