• 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...

Key Points Clot contraction has 3 phases differentially affected by platelet and fibrin mechanics, RBC compaction, and various blood components. A new dynamic quantitative clot contraction assay can reveal novel aspects of formation and evolution of hemostatic clots and thrombi.

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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.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.