Background Thrombosis and inflammation may contribute to the risk of death and complications among patients with coronavirus disease 2019 (Covid-19). We hypothesized that therapeutic-dose anticoagulation may improve outcomes in noncritically ill patients who are hospitalized with Covid-19. Methods In this open-label, adaptive, multiplatform, controlled trial, we randomly assigned patients who were hospitalized with Covid-19 and who were not critically ill (which was defined as an absence of critical care–level organ support at enrollment) to receive pragmatically defined regimens of either therapeutic-dose anticoagulation with heparin or usual-care pharmacologic thromboprophylaxis. The primary outcome was organ support–free days, evaluated on an ordinal scale that combined in-hospital death (assigned a value of −1) and the number of days free of cardiovascular or respiratory organ support up to day 21 among patients who survived to hospital discharge. This outcome was evaluated with the use of a Bayesian statistical model for all patients and according to the baseline d -dimer level. Results The trial was stopped when prespecified criteria for the superiority of therapeutic-dose anticoagulation were met. Among 2219 patients in the final analysis, the probability that therapeutic-dose anticoagulation increased organ support–free days as compared with usual-care thromboprophylaxis was 98.6% (adjusted odds ratio, 1.27; 95% credible interval, 1.03 to 1.58). The adjusted absolute between-group difference in survival until hospital discharge without organ support favoring therapeutic-dose anticoagulation was 4.0 percentage points (95% credible interval, 0.5 to 7.2). The final probability of the superiority of therapeutic-dose anticoagulation over usual-care thromboprophylaxis was 97.3% in the high d -dimer cohort, 92.9% in the low d -dimer cohort, and 97.3% in the unknown d -dimer cohort. Major bleeding occurred in 1.9% of the patients receiving therapeutic-dose anticoagulation and in 0.9% of those receiving thromboprophylaxis. Conclusions In noncritically ill patients with Covid-19, an initial strategy of therapeutic-dose anticoagulation with heparin increased the probability of survival to hospital discharge with reduced use of cardiovascular or respiratory organ support as compared with usual-care thromboprophylaxis. (ATTACC, ACTIV-4a, and REMAP-CAP ClinicalTrials.gov numbers, NCT04372589 , NCT04505774 , NCT04359277 , and NCT02735707 .)

Low doses of ER niacin (1000 or 1500 mg/d) are a treatment option for dyslipidemia in patients with type 2 diabetes.

Thrombi contains electropositive patches at opposite poles of the molecule which represent potential exosites for the binding of macromolecular ligands. The Thrombin is a critical regulator of hemostasis and is an important target for anticoagulant therapy in thromboembolic disorders. The principal anticoagulant drug for the treatment of acute thrombosis is heparin, a glycosaminoglycan that catalyzes the inhibition of thrombin and other serine proteases by the serpin (serine protease inhibitor) antithrombin III (ATIII) (1). Endogenous glycosaminoglycans may regulate thrombin by a similar mechanism (2). The inhibition of thrombin by ATIII is a two-step process in which an initial, reversible complex is converted to a stable ATIII-thrombin complex in a first-order reaction. Heparin increases the affinity of the initial complex by nearly three orders of magnitude (3). The kinetics of inhibition are consistent with binding of both inhibitor and protease to heparin in a template fashion. In contrast to the inhibition of factor Xa, heparin-induced conformational change in ATIII contributes little to the acceleration of thrombin inhibition (4). Because heparin binds with higher affinity to ATIII than to thrombin, inhibition proceeds mainly through the interaction of ATIII-heparin complexes with free thrombin (5). Thrombin-heparin binding appears to be predominantly electrostatic, involving at least five ionic interactions. Thrombin does not require a specific heparin oligosaccharide sequence but interacts with relatively nonspecific sites approximately three disaccharides in length, with an intrinsic Kd of 5-10 pM (6).The surface of thrombin is characterized by two large electropositive patches at opposite poles of the molecule which represent potential exosites for the binding of negatively charged glycosaminoglycan ligands (7). The fibrin(ogen) recognition site, or anion-binding exosite I, is located in a region suitable for binding amino acid residues on the carboxyl side of the scissile bond of thrombin substrates (7). Exosite I contributes to the unique specificity of thrombin, binding not only substrates such as fibrinogen (8, 9) and the thrombin receptor (10) but also cofactors such as thrombomodulin (9) and inhibitors such as heparin cofactor 11 (11), hirudin (12), and single-stranded DNA aptamers (13). The opposing electropositive surface patch, anion-binding exosite II, consists of the carboxyl-terminal a-helix of the protease and portions of adjacent surface loops. The kringle 2 domain of prothrombin forms a noncovalent complex with thrombin by interacting with exosite I (14), indicating that this exosite is not accessible in prothrombin. Upon activation to thrombin, kringle 2 dissociates and exosite II is exposed.After activation to a-thrombin, no function has been demonstrated directly for anion-binding exosite II. Heparin protects lysine residues (Lys-174 and Lys-252) within exosite II from chemical modification, suggesting that this region may contain a heparin-binding site (15). Furthermore, t...

Summary. To analyse primary haemostasis in the zebra®sh we have identi®ed and characterized the zebra®sh thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebra®sh blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/ aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebra®sh treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebra®sh thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebra®sh is a relevant model for mammalian haemostasis and thrombosis.

Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen-and thrombomodulinbinding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had ==2.5-fold increased protein C-activating activity but only =17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogenclotting activity but only -7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin-and fibrinogen-binding sites on thrombin may overlap but are not identical.

Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IXa with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). These mutations only had modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by factor IXa. Likewise, factor IXa H92A and K241A showed factor IXa-factor VIIIa affinity similar to factor IXa wild type (WT). In contrast, factor IXa R170A demonstrated a 4-fold increase in apparent factor IXa-factor VIIIa affinity and dramatically increased coagulant activity relative to factor IXa WT. Factor IXa R233A demonstrated a 2.5-fold decrease in cofactor affinity and reduced ability to stabilize cofactor half-life relative to wild type, suggesting that interaction with the factor VIIIa A2 domain was disrupted. Markedly (R233A) or moderately (H92A, R170A, K241A) reduced binding to immobilized LMWH was observed for the mutant proteases. Solution competition demonstrated that the EC(50) for LMWH was increased less than 2-fold for factor IXa H92A and K241A but over 3.5-fold for factor IXa R170A, indicating that relative heparin affinity was WT > H92A/K241A > R170A >> R233A. Kinetic analysis of intrinsic tenase inhibition demonstrated that relative affinity for LMWH was WT > K241A > H92A > R170A >> R233A, correlating with heparin affinity. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite in the factor IXa protease domain, which disrupts interaction with the factor VIIIa A2 domain.

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