Search

BACKGROUND Plasma thawed and stored at 1 to 6°C for up to 5 days (thawed plasma [TP]) provides rapid availability in emergencies and reduces plasma waste, but it carries risks of coagulation factor loss or activation, bacterial outgrowth, and viral contamination. We characterized changes in amotosalen/ultraviolet A (UVA) light pathogen-reduced, fresh-frozen plasma (FFP) and plasma frozen within 24 hours (PF24) with post-thaw storage. STUDY DESIGN AND METHODS Amotosalen/UVA light-treated FFP and PF24 were thawed after approximately 3 to more than 12 months of frozen storage and held at 1 to 6°C for 5 days. Global assessments of coagulation and hemostatic, antithrombotic, and activation markers indicative of function were assessed. RESULTS Day 5, thawed amotosalen/UVA light-treated FFP and PF24 contained levels of Factors II, V, VIII, IX, X, von Willebrand factor ristocetin cofactor (vWF:RCo), fibrinogen, antithrombin III (ATIII), protein C, and protein S similar to the levels measured in Day 5 TP, as described in the Circular of Information. Thrombin generation was robust on Day 5 (amotosalen/UVA: FFP = 1866 ± 402 nM/minute; PF24 = 1800 ± 277 nM/minute). Most factor activities on Day 5, including von Willebrand factor-cleaving protease (ADAMTS-13), were more than 90% of Day 0 values, except for known labile Factors V and VIII and protein S. All units contained greater than 0.4 IU/mL protein S and α2 plasmin inhibitor on Day 5. Global functional indices, including thrombin-antithrombin complexes, nonactivated thromboplastin time, and thrombin-generation peak height, did not indicate activation of the coagulation cascade, although isolated units showed raised levels of Factor VIIa and Complement 3a. CONCLUSION Amotosalen/UVA light-treated FFP and PF24 demonstrated retention of procoagulant and antithrombotic activity after 5 days post-thaw storage at 1 to 6°C.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Plasma thawed and stored at 1 to 6Amotosalen/UVA light-treated FFP and PF24 were thawed after approximately 3 to more than 12 months of frozen storage and held at 1 to 6Day 5, thawed amotosalen/UVA light-treated FFP and PF24 contained levels of Factors II, V, VIII, IX, X, von Willebrand factor ristocetin cofactor (vWF:RCo), fibrinogen, antithrombin III (ATIII), protein C, and protein S similar to the levels measured in Day 5 TP, as described in the Circular of Information. Thrombin generation was robust on Day 5 (amotosalen/UVA: FFP = 1866 ± 402 nM/minute; PF24 = 1800 ± 277 nM/minute). Most factor activities on Day 5, including von Willebrand factor-cleaving protease (ADAMTS-13), were more than 90% of Day 0 values, except for known labile Factors V and VIII and protein S. All units contained greater than 0.4 IU/mL protein S and α2 plasmin inhibitor on Day 5. Global functional indices, including thrombin-antithrombin complexes, nonactivated thromboplastin time, and thrombin-generation peak height, did not indicate activation of the coagulation cascade, although isolated units showed raised levels of Factor VIIa and Complement 3a.Amotosalen/UVA light-treated FFP and PF24 demonstrated retention of procoagulant and antithrombotic activity after 5 days post-thaw storage at 1 to 6

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3 −/− and Par4 +/− mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12 +/− mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3 −/− mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl 3 injury. Par4 +/− and P2ry12 +/− mice showed partial protection. Increasing FeCl 3 injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3 −/− : P2ry12 +/− mice, restored it. Par4 −/− mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that ( i ) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; ( ii ) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and ( iii ) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.

Coagulation factor XIa is a candidate target for anticoagulants that better separate antithrombotic efficacy from bleeding risk. We report a co-crystal structure of the FXIa protease domain with DEF, a human monoclonal antibody that blocks FXIa function and prevents thrombosis in animal models without detectable increased bleeding. The light chain of DEF occludes the FXIa S1 subsite and active site, while the heavy chain provides electrostatic interactions with the surface of FXIa. The structure accounts for the specificity of DEF for FXIa over its zymogen and related proteases, its active-site-dependent binding, and its ability to inhibit substrate cleavage. The inactive FXIa protease domain used to obtain the DEF-FXIa crystal structure reversed anticoagulant activity of DEF in plasma and invivo and the activity of a small-molecule FXIa active-site inhibitor invitro. DEF and this reversal agent for FXIa active-site inhibitors may help support clinical development of FXIa-targeting anticoagulants.

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements., Functional imaging of proteolytic activity is an emerging strategy to guide patient diagnosis and monitor clinical outcome. Here the authors present a peptide-based probe to detect and localize thrombin activity ex vivo and non-invasively in mouse models of wounding and pulmonary thrombosis.

Fibrin(ogen) is central to hemostasis and thrombosis and also contributes to multiple physiologic and pathologic processes beyond coagulation. However, the precise contribution of soluble fibrinogen vs insoluble fibrin matrices to vascular integrity, tissue repair, inflammation, and disease has been undefined and unapproachable. To establish the means to distinguish fibrinogen- and fibrin-dependent processes in vivo, Fib(AEK) mice were generated that carry normal levels of circulating fibrinogen but lack the capacity for fibrin polymer formation due to a germ-line mutation in the Aα chain thrombin cleavage site. Homozygous Fib(AEK) mice developed to term and exhibited postnatal survival superior to that of fibrinogen-deficient mice. Unlike fibrinogen-deficient mice, platelet-rich plasma from Fib(AEK) mice supported normal platelet aggregation in vitro, highlighting that fibrinogen(AEK) retains the functional capacity to support interactions with platelets. Thrombin failed to release fibrinopeptide-A from fibrinogen(AEK) and failed to induce polymer formation with Fib(AEK) plasma or purified fibrinogen(AEK) in 37°C mixtures regardless of incubation time. Fib(AEK) mice displayed both an absence of fibrin polymer formation following liver injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi following FeCl3 injury of carotid arteries. Fib(AEK) mice exhibited a profound impediment in Staphylococcus aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet Fib(AEK) mice displayed a significant infection dose-dependent survival advantage over fibrinogen-deficient mice following peritonitis challenge. Collectively, these findings establish for the first time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while simultaneously highlighting biologically meaningful contributions and functions of the soluble molecule.

Contexte Le cryoprecipite (CPT) est utilise dans certains pays comme apport de fibrinogene chez les patients avec une hemorragie majeure lors d’une chirurgie cardiaque ou d’un traumatisme. La preparation de CPT a partir de melanges de plasma traite par Intercept™ (MPFC-IA) reduit le risque infectieux et permet potentiellement une conservation a 22 °C. Objectif Evaluer la conservation du CPT issu de MPFC-IA (CPT-IA) a temperature ambiante apres decongelation. Methode Dix unites de plasma de groupe A issues de sang total ont ete melangees et divisees en 3 unites (∼ 640 mL chacune) traitees par Intercept™ et 3 unites de controle non traitees (∼ 325 mL chacune). Le MPFC-IA a ete transfere dans une poche de 800 mL, congele a −30 °C, decongele a 4 °C pendant 24 heures et centrifuge a 4200 g, 12′ pour concentrer le CPT-IA puis congele avec ∼ 60 mL de surnageant a −30 °C. Le CPT controle (CPT-C) a ete prepare de maniere equivalente. Apres 4 jours de stockage a −30 °C, les CPT-IA et CPT-C ont ete decongeles a 37 °C et conserves a 22 °C. Les concentrations de fibrinogene (FI), FVIII, FXIII et VWF(Ag) ont ete mesurees a differentes periodes. Resultats La concentration de FI est restee stable dans les CPT-IA et CPT-C alors que le FVIII a montre une baisse reguliere (voir Tableau 1 ). Les niveaux de FXIII et de vWF sont restes stables jusqu’a 120 h. La fonction hemostatique du cryoprecipite en utilisant le ROTEM et le test de generation de thrombine a ete verifiee. Conclusion La conservation du cryoprecipite Intercept™ decongele jusqu’a 120 h a 22 °C a peu d’impact sur les facteurs de coagulation critiques. Ces donnees soutiennent une evaluation plus approfondie de ce produit.

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1–deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments. In the prevailing model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue. Acting via the β(3)-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes, whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.

See also Gardiner EE. A GPIb-IX-V complex signaling environment. This issue, pp 1075–6. Summary. Background: Circulating platelets are initially recruited at the site of vessel injury by von Willebrand factor (VWF) immobilized on collagen fibers. This process, mediated by the GPIb–V–IX complex, is accompanied by specific intracellular signaling leading to reorganization of the platelet actin cytoskeleton and extension of filopodia. Objectives/methods: To evaluate the GPIbα and GPIbβ intracellular domains contribution to this signaling, we generated Chinese hamster ovary (CHO) cells expressing a GPIb–IX complex with mutant forms of the two subunits and we measured their ability to extend filopodia upon adhesion on a VWF matrix. Results: Complete intracellular deletion or elimination of the filamin or the 14-3-3ζ binding sites in GPIbα did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu150–Arg160) or central (Ala159–Pro170) intracellular segment of GPIbβ resulted in a 21% and 23% reduction in the number of cells extending filopodia, respectively. This occurred without decreasing adhesion efficiency or GPIb–IX association with filamin A or 14-3-3ζ. Alanine scanning mutagenesis of the Leu150–Pro170 segment identified Arg164, Leu165, Leu167, Thr168 and Pro170 as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser166 PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbβ subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock-in mice expressing a GPIbβ intracellular deletion mutant. Conclusions: Altogether, our results strongly support participation of GPIbβ and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.

To cite this article: Mangin PH, Receveur N, Wurtz V, David T, Gachet C, Lanza F. Identification of five novel 14-3-3 isoforms interacting with the GPIbIX complex in platelets. J Thromb Haemost 2009; 7: 15505. Summary. Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)IbIX complex initiates a signaling cascade leading to integrin aIIbb3 activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3f with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective :T he aim of our study was to determine whether other members of the 14-33 family bind to GPIbIX. Results: In this study, western blot analyses showed that platelets also contain the 14-3-3b, 14-3-3c, 14-3-3e ,1 4-3-3g and 14-3-3h isoforms, but lack 14-3-3r. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3f ,b ind to GPIbIX. In addition, their interaction was found to critically require the same GPIba domains (580590 and 605610) already identified as essential for 14-3-3f binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbb. In contrast, deletion or mutation of the GPIbb intracytoplasmic tail did not affect the interaction of GPIbIX with the 14-3-3 isoforms, questioning the importance of this domain. Conclusions: Our study suggests that, to inhibit GPIb-induced integrin aIIbb3 activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Summary. Background: Interaction between the platelet glycoprotein (GP)Ib-V-IX complex and von Willebrand factor (VWF) is critical for initiating platelet-vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin αIIbβ3 activation. Objective: A cell-penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbα subunit in VWF/GPIb-V-IX-dependent adhesion and activation. Methods: Peptides of 11–13 amino acids, covering the 557–610 region, were coupled to a nine-arginine permeating tag (R9) and the effects of their cell entry on VWF-dependent responses were analyzed. Results: The R9α557 peptide corresponding to the 557–569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9α557 on GPIb VWF-dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb-IX complex were incubated with R9α557. Conclusion: This study performed in intact platelets suggests a functional role of the 557–569 domain of GPIbα in controlling VWF-dependent adhesion and signaling.

Objective— The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIbβ subunit. Methods and Results— Complete deletion (GPIbβ −/− ) or an intracellular truncation (GPIbβΔIC −/− ) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIbβΔIC −/− . Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl 3 -induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIbβ −/− mice, while a more modest effect was observed in GPIbβΔIC −/− animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. Conclusions— This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.

Abstract 1111 Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott Syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F-knockout mice that exhibit bleeding defects associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity, and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. TMEM16F channels permeate both monovalent and divalent cations including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a novel Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity. Disclosures: No relevant conflicts of interest to declare.

Endothelial and hematopoietic tissue factor are unnecessary in two mouse models of hemostasis and thrombosis Tissue Factor (TF), in combination with Factor VII, activates the extrinsic coagulation pathway leading to thrombin generation and clot formation. TF is expressed by epithelial, smooth muscle and perivascular cells; in addition endothelial and hematopoietic cells can express TF, especially in response to inflammatory stimuli. Despite extensive studies the exact cellular source of TF during clot initiation and propagation has not yet been clearly identified, and the importance of endothelial or hematopoietic TF in hemostasis and thrombosis is unknown. To address the latter, we measured bleeding time and arterial thrombus formation in TF flox/flox: Tie-2p/e Cre conditional mice, in which endothelial and circulating TF expression is absent. We observed that TF deficiency in the endothelial and hematopoietic compartment was not associated with defective hemostasis. In a FeCl3- induced carotid injury model, in contrast to PAR4 null mice, clotting in TF conditional mice did not significantly differ from that of wild type littermates. This study suggests that TF derived from endothelial or blood cells is not necessary for hemostasis and thrombosis in the models studied. Figure Figure