• In contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface.• Polyhedrocytes form an impermeable seal to stem bleeding and help prevent vascular obstruction but confer resistance to fibrinolysis.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. (Blood. 2014;123(10):1596-1603 IntroductionBlood clotting is a necessary part of hemostasis in which platelets aggregate to form a temporary sealant and fibrinogen is converted to a network of fibrin polymers to stem bleeding, yet both of these processes are also linked to thrombosis. [1][2][3][4] The resulting viscoelastic gel then contracts through the action of cytoplasmic motility proteins inside platelets, such that fluid (serum) is expelled, a process called clot contraction or retraction. Clots made from platelet-rich plasma (PRP) generate a bulk contractile force that begins shortly after the clot is formed and increases over minutes to hours to a maximum of about 1500 to 4500 dyn/cm 2 . 5,6 The function of clot contraction is not fully known, but it appears to reinforce hemostasis by forming a seal, promote wound healing by approximating the edges, and restore blood flow by decreasing the area obstructed by intravascular clots. [6][7][8] Although erythrocytes are a major component of blood clots, little is known about their participation in clot contraction. Historically, the presence of erythrocytes in contracted blood clots has been recognized and sometimes exploited; for example, during the time of medical bloodletting, the size of the contracted clot from blood removed from the patient was used as a measure of erythrocyte mass to determine when the procedure should cease. 6 Moreover, erythrocyte...

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.

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. IntroductionPlatelet factor 4 (PF4) is a cationic chemokine with high affinity for unfractionated heparin (UFH) and other large, negatively charged molecules. 1 PF4 is stored in platelet ␣-granules, released upon activation, when it then binds rapidly to glycosaminoglycan (GAG) side chains expressed on the surface of platelets 2 and other vascular cells, with little remaining free in the circulation. 3 Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize complexes of human (h) PF4 with heparin or other GAGs. 4,5 In solution, formation of antigenic complexes between PF4 and heparin is critically dependent on their molar ratio, with loss of antibody binding when the optimal ratio is disrupted by an excess of either component. 6,7 Antigen formation on the platelet surface also follows a bell-shaped curve as PF4 concentration is increased, with maximal binding of antibody seen at an exogenous PF4 concentration of 50 g/mL. 8 Chondroitin sulfates (CSs) are the predominant GAG side chains expressed on platelets. 9,10 We have shown that the binding of the HIT-like monoclonal antibody KKO 11 to platelets is abrogated by chondroitinase ABC, 8 indicating that HIT antibodies bind to PF4/CS complexes on this cell type. Therapeutic concentrations of UFH disrupt antibody binding in part by eluting PF4 from the platelet surface, which reduces formation of antigenic complexes and the potential for platelet activation 8 through platelet Fc␥RIIA. 12 Thus, variation in the expression of platelet-derived PF4 (or CS) might help to explain why only a small percentage of patients who generate antibodies to PF4/UFH develop HIT. 13 Although it has been generally accepte...

Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the ‘closed' end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the ‘open' end of the tetramer. Fondaparinux and KKO thereby collaborate to ‘stabilize' the ternary pathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. The atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.

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.

• The procoagulant nature of HIT can be simulated in a microfluidic model using human blood and its components.• PF4/glycosaminoglycans/ immunoglobulin G complexes activate monocytes through FcgRIIA to generate TF and thrombin, leading to coated platelets in HIT.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)gRIIA 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 FcgRIIA, 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 FcgRIIA 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 FcgRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk. (Blood. 2016;127(4):464-472) IntroductionHeparin-induced thrombocytopenia (HIT) is an iatrogenic, immunemediated disorder characterized by antibodies that recognize complexes between the platelet chemokine platelet factor 4 (PF4, CXCL4) and heparin or cell surface glycosaminoglycans (GAGs). 1,2 It is estimated that up to 50% of patients with HIT develop thrombosis that might be limb-and/or life-threatening. [3][4][5] Even with early recognition, cessation of heparin, and institution of alternative forms of anticoagulation, recurrent thromboembolic complications may occur and 10% to 20% of patients go on to amputation and/or death. 6 Thus, there is a need for a better understanding of the pathogenesis of HIT and to determine how this information can be used to mitigate the risk of thrombosis.Thrombocytopenia and thrombosis in HIT have been attributed to binding of PF4/heparin/immunoglobulin G (IgG) immune-complexes to the platelets through the IgG fragment crystallizable (Fc) region, which activates platelets through their immunoreceptor tyrosine-based activation motif (ITAM) receptor, FcgRIIA. 7,8 However, monocytes, endothelial cells, and other cell types might also be activated by these immune complexes and contribute to the underlying pathology, 9 but their contribution to the process is less well characterized. Indeed, recent evidence suggests that thrombosis in HIT is initiated by binding of pathogenic antibodies to antigenic complexes of PF4 and GAGs expressed by the endothelium as well as circulating cells, includi...

• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)

Several CGHC motif-containing disulfide isomerases support thrombosis. We here report that endoplasmic reticulum protein 72 (ERp72), with 3 CGHC redox-active sites (a, a, and a'), supports thrombosis. We generated a new conditional knockout mouse model and found that Tie2-Cre/ERp72 mice with blood and endothelial cells lacking ERp72 had prolonged tail bleeding times and decreased platelet accumulation in laser-induced cremaster arteriole injury and FeCl-induced mesenteric arterial injury. Fibrin deposition was decreased in the laser injury model. Both platelet and fibrin accumulation defects were fully rescued by infusion of recombinant ERp72 containing functional a and a' CGHC motifs (ERp72(oo-ss-ss)). Infusion of ERp72 containing inactivated a and a' CGHC motifs (ERp72(ss-oo-oo)) inhibited platelet accumulation and fibrin deposition in wild-type mice. Infusion of ERp72(oo-ss-ss) into β3-null mice increased fibrin deposition in the absence of platelets. ERp72-null platelets had defective aggregation, JON/A binding, P-selectin expression, and adenosine triphosphate (ATP) secretion. The aggregation and ATP secretion defects were fully rescued by ERp72(oo-ss-ss) but partially rescued by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). Aggregation and ATP secretion of human platelets was potentiated by ERp72(oo-ss-ss) but inhibited by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). These data suggest that both the a and a' active sites are required for platelet function. ERp72 bound poorly to β3-null mouse platelets, and the addition of ERp72(oo-ss-ss) to human platelets generated thiols in αIIbβ3, suggesting a direct interaction of ERp72 with αIIbβ3. Defective aggregation of ERp72-null platelets was recovered by ERp72, but not other thiol isomerases. In summary, ERp72 plays a critical role in platelet function and coagulation through the a and a' CGHC motifs.