Lubica Rauova

Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a life- and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation.

Adverse effects of intravenous immunoglobulin therapy in 56 patients with autoimmune diseases.

Objective: To test the adverse effects and viral safety of intravenous immunoglobulin (IVIg) use in autoimmune diseases. Methods: Fifty-six patients with various autoimmune diseases who were treated with one to six IVIg courses were evaluated for the presence of adverse effects following IVIg therapy and were screened before and after the treatment for the presence of serum human immunodeficiency virus antibodies, hepatitis C virus antibodies, and hepatitis B surface antigen. Results: Among the 56 patients, 20 (36%) had at least one adverse effect following at least one of the treatment courses. These included headache, low-grade fever, chills, anemia, low-back pain, transient hypotension, nausea, intensified perspiration, and superficial and deep vein thromboses. Whereas the presence of adverse effect to IVIg was unrelated to either the clinical response to the treatment or to the nature of the autoimmune disease, the occurrence of an adverse effect in the first treatment course was significantly associated with a greater chance for an adverse effect in the subsequent courses. No transmission of any of the three viral agents examined could be detected. Conclusions: Although IVIg use in autoimmune diseases is associated with adverse effects in about one third of the patients, these effects are usually mild and transient. Patients who develop adverse effects during the first treatment course may be at increased risk of adverse effects during the subsequent IVIg courses.

Infusion of mature megakaryocytes into mice yields functional platelets

Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes, remains incompletely understood. Prior studies suggest that megakaryocytes shed platelets in the pulmonary vasculature. To better understand thrombopoiesis and to develop a potential platelet transfusion strategy that is not dependent upon donors, of which there remains a shortage, we examined whether megakaryocytes infused into mice shed platelets. Infused megakaryocytes led to clinically relevant increases in platelet numbers. The released platelets were normal in size, displayed appropriate surface markers, and had a near-normal circulating half-life. The functionality of the donor-derived platelets was also demonstrated in vivo. The infused megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers.

Role of the platelet chemokine platelet factor 4 (PF4) in hemostasis and thrombosis.

Chemokines are a family of small proteins that have significant roles in inflammation, angiogenesis and cellular homing. Since inflammation and hemostasis/thrombosis have multiple overlapping roles and pathways, one could expect that some chemokines would also have biologically significant roles in hemostasis/thrombosis as well. This would especially be true for chemokines that are localized solely or predominantly within platelets and released in large amounts at sites of platelet activation such as platelet factor 4 (PF4, CXCL4) and its closely related chemokine, platelet basic protein (PBP, CXCL7). Our group and others have clearly demonstrated an in vivo role for PF4 in hemostasis/thrombosis, but not for PBP, which in contrast has clear proinflammatory properties. This review will focus on PF4 and its potential roles in hemostasis/thrombosis and the underlying pathways by which PF4 may be especially important in such pathologic thrombotic states as heparin-induced thrombocytopenia (HIT) and septic shock.

High-dose intravenous immunoglobulins for lupus nephritis — a salvage immunomodulation

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with a great diversity of clinical manifestations which is difficult to manage. IVIGs represent promising immunoregulatory agents with the ability to control SLE without subsequent predisposition to infectious complications. Despite the implied risk of developing renal failure due to IVIG, considerable beneficial effects on lupus nephritis are reported. In this review, the clinical and adverse effects, and machanism of action, with special emphasis on modulation, of idiotypic network is discussed.

Dynamic antibody-binding properties in the pathogenesis of HIT

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.

Platelet‐delivered factor VIII provides limited resistance to anti‐factor VIII inhibitors

Summary.  Background: Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti‐FVIII inhibitors. Objective: We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIIInull mouse in the presence of circulating inhibitors. Methods: FVIIInull mice that were transgenic for pFVIII (pFVIII/FVIIInull) were compared with FVIIInull mice receiving infused FVIII in a FeCl3 carotid injury model in the presence of anti‐FVIII inhibitors. Results: After injury, pFVIII/FVIIInull mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia. Conclusion: In FVIIInull mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of ∼6‐fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail‐clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail‐vein exsanguination model to low levels of pFVIII.

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation.