Andrew J. Gale

Activated protein c variants with normal cytoprotective activity but reduced anticoagulant activity

Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APCs less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APCs anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells.

Activated protein C

Summary.  Protein C is a vitamin K‐dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down‐regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti‐inflammatory and anti‐apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor‐1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC’s direct effects on various cell types, it is clear that APC’s molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.

Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.

Antibodies confer humoral immunity but can also be harmful when they target an autoantigen, alloantigen, allergen, or biotherapeutic. New strategies are needed for antigen-specific suppression of undesired antibody responses, particularly to T cell-dependent protein antigens, because they elicit T cell help. Here we show that liposomal nanoparticles, displaying both antigen and glycan ligands of the inhibitory coreceptor CD22, induce a tolerogenic program that selectively causes apoptosis in mouse and human B cells. These SIGLEC-engaging tolerance-inducing antigenic liposomes (STALs, where SIGLEC is defined as sialic acid-binding Ig-like lectin) induced robust antigen-specific tolerance to protein antigens in mice, preventing subsequent immune response to challenge with the same antigen. Since development of inhibitory antibodies to FVIII is a serious problem in treatment of hemophilia A patients, we investigated the potential of this approach for inducing tolerance to FVIII in a hemophilia mouse model. STALs prevented formation of inhibitory FVIII antibodies, allowing for effective administration of FVIII to hemophilia mice to prevent bleeding. These findings suggest that STALs could be used to eliminate or prevent harmful B cell-mediated immune responses.

Cretaceous Extinctions: Multiple Causes

![Figure][1] Deccan plateau basalts. Lava from Deccan volcanism formed distinct layering. CREDIT: GSFC/NASA In the Review “The Chicxulub Asteroid Impact and Mass Extinction at the Cretaceous-Paleogene boundary” (P. Schulte et al. , 5 March, p. [1214][2]), the terminal Cretaceous

Update on Tumor Cell Procoagulant Factors

Tumor cells produce tissue factor, cancer procoagulant, plasminogen activators and other factors that interact with the coagulation system, the fibrinolytic system and vascular or blood cells such that they can upset the normal homeostasis and balance between activation and inhibition of the coagulation and fibrinolytic systems. These activities play a role in tumor cell growth and metastasis, vascular wall function, and hemostasis. Proteases and their inhibitors are intimately involved in all aspects of the hemostatic, cell proliferation and cellular signalling systems. This review provides a brief examination of recent observations in this complex interaction of cellular and hemostatic factors.

Molecular Characterization of an Extended Binding Site for Coagulation Factor Va in the Positive Exosite of Activated Protein C

The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys193, Arg229, and Arg230. In addition, four residues made significant minor contributions to FVa interactions: Lys191, Lys192, Asp214, and Glu215. All of these residues primarily contribute to APC cleavage at Arg506 in FVa and play a small role in the interaction of APC with the Arg306 cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg506 on FVa.

An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa.

Summary.  The blood coagulation disorder, hemophilia A, is caused by deficiency of coagulation factor (F)VIII. Hemophilia A is now treated by infusions of pure FVIII, but the activity of FVIII is limited because it is unstable following activation by thrombin. This instability of activated FVIII is the result of dissociation of the A2 subunit. To obtain increased stability in FVIIIa, a disulfide bond between the A2 domain and the A3 domain, preventing A2 subunit dissociation, has been engineered. Structural analysis of the FVIII A domain homology model allowed us to identify residues 664 and 1826 as a potential disulfide bond pair. A FVIII mutant containing Cys664 and Cys1826 was produced and purified (C664‐C1826 FVIII). Immunoblotting showed that a disulfide bond did form to link covalently the A2 and the A3 domains. Following activation of the recombinant C664‐C1826 FVIII by thrombin, the mutant FVIIIa had increased stability and retained more than 90% of its clotting activity at a time at which wild‐type FVIIIa lost more than 90% of its activity. This remarkably stable C664‐C1826 FVIIIa provides a unique approach for studies of the cofactor activity of FVIIIa and also for new, improved therapy for hemophilia A.

Continuing Education Course #2: Current Understanding of Hemostasis:

The goal of this review is to briefly summarize the two primary pathways of hemostasis, primary hemostasis and secondary hemostasis, as well as to summarize anticoagulant mechanisms and fibrinolysis. In addition, this review will discuss pathologies of hemostasis and the mechanisms of the various drugs that are available to impact these pathways to prevent either thrombosis or bleeding. While many of the main drugs that are used to treat disorders of hemostasis have been used for decades, greater understanding of hemostasis has led to development of various new drugs that have come onto the market recently or are close to coming onto the market. Thus, improved understanding of hemostasis continues to lead to benefits for patients.

Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C

Procoagulant factor Va (FVa) is inactivated via limited proteolysis at three Arg residues in the A2 domain by the anticoagulant serine protease, activated protein C (APC). Cleavage by APC at Arg306 in FVa causes dissociation of the A2 domain from the heterotrimeric A1:A2:A3 structure and complete loss of procoagulant activity. To help distinguish inactivation mechanisms involving A2 domain dissociation from inactivation mechanisms involving unfavorable changes in factor Xa (FXa) affinity, we used our FVa homology model to engineer recombinant FVa mutants containing an interdomain disulfide bond (Cys609‐Cys1691) between the A2 and A3 domains (A2‐SS‐A3 mutants) in addition to cleavage site mutations, Arg506Gln and Arg679Gln. SDS‐PAGE analysis showed that the disulfide bond in A2‐SS‐A3 mutants prevented dissociation of the A2 domain. In the absence of A2 domain dissociation from the A1:A2:A3 trimer, APC cleavage at Arg306 alone caused a sevenfold decrease in affinity for FXa, whereas APC cleavages at Arg306, Arg506, and Arg679 caused a 70‐fold decrease in affinity for FXa and a 10‐fold decrease in the kcat of the prothrombinase complex for prothrombin without any effect on the apparent Km for prothrombin. Therefore, for FVa inactivation by APC, dissociation of the A2 domain may provide only a modest final step, whereas the critical events are the cleavages at Arg506 and Arg306, which effectively inactivate FVa before A2 dissociation can take place. Nonetheless, for FVa Leiden (Gln506‐FVa) inactivation by APC, A2 domain dissociation may become mechanistically important, depending on the ambient FXa concentration.

Intrinsic stability and functional properties of disulfide bond‐stabilized coagulation factor VIIIa variants

Summary.  Background: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664–C1826) in recombinant FVIII that formed a disulfide bond cross‐linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. Aims: Here, we characterize the functional parameters of C664–C1828 FVIII and of a new disulfide bond‐stabilized FVIII (C662–C1828 FVIII). Methods: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half‐life of disulfide bond‐stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. Results: Both disulfide bond‐stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662–C1828 FVIIIa had normal activity while C664–C1826 FVIIIa had reduced activity. Both C664–C1826 FVIIIa and C662–C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. Conclusion: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.