Frank C. Church

Serpins in thrombosis, hemostasis and fibrinolysis.

Summary.  Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin–protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure–function relationships is imperative for the development of state‐of‐the‐art pharmaceuticals for the selective fine‐tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z‐dependent protease inhibitor, α1‐protease inhibitor, protein C inhibitor, α2‐antiplasmin and plasminogen activator inhibitor‐1. We focus on the biological function, the important structural elements, their known non‐hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique β-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin β-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin–HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central β-sheet of HCII.

Protein C Inhibitor Is a Potent Inhibitor of the Thrombin-Thrombomodulin Complex

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI ≈140-fold (k = 2.4 × 10 in the presence of TM compared to 1.7 × 10M s in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI ≈59-fold (k = 1.0 × 10M s). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k = 6.3 × 10M s), but TM 4-6 enhances inhibition by this mutant ≈9-fold (k = 5.8 × 10M s) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

An o-phthalaldehyde spectrophotometric assay for proteinases☆

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.

Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans.

Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer.

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice

Epidemiologic studies have correlated elevated plasma fibrinogen (hyperfibrinogenemia) with risk of cardiovascular disease and arterial and venous thrombosis. However, it is unknown whether hyperfibrinogenemia is merely a biomarker of the proinflammatory disease state or is a causative mechanism in the etiology. We raised plasma fibrinogen levels in mice via intravenous infusion and induced thrombosis by ferric chloride application to the carotid artery (high shear) or saphenous vein (lower shear); hyperfibrinogenemia significantly shortened the time to occlusion in both models. Using immunohistochemistry, turbidity, confocal microscopy, and elastometry of clots produced in cell and tissue factor-initiated models of thrombosis, we show that hyperfibrinogenemia increased thrombus fibrin content, promoted faster fibrin formation, and increased fibrin network density, strength, and stability. Hyperfibrinogenemia also increased thrombus resistance to tenecteplase-induced thrombolysis in vivo. These data indicate that hyperfibrinogenemia directly promotes thrombosis and thrombolysis resistance and does so via enhanced fibrin formation and stability. These findings strongly suggest a causative role for hyperfibrinogenemia in acute thrombosis and have significant implications for thrombolytic therapy. Plasma fibrinogen levels may be used to identify patients at risk for thrombosis and inform thrombolytic administration for treating acute thrombosis/thromboembolism.

Plasminogen Activator Inhibitor-1 and -3 Increase Cell Adhesion and Motility of MDA-MB-435 Breast Cancer Cells*

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of α2, α3, α4, α5, and β1 integrin subunits. Additionally, blocking antibodies to β1integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.

Chemistry and biology of serpins

Introduction: Serpins: From the Way It Was to the Way It Is J. Travis. Serpins: A Mechanistic Class of Their Own S.R. Stone et al.. Coagulation: Antithrombin--A Bloody Important Serpin I. Bjork, S.T. Olson. Heparin Cofactor II D.M. Tollefsen. Neurobiology and Cancer: Regulation of Neurons and Astrocytes by Thrombin and Protease Nexin--l: Relationship to Brain Injury D.D. Cunningham, F.M. Donovan. Maspin: A Tumor Suppressing Serpin R. Sager et al.. Fibrinolysis: The Role of Reactive--Center Loop Mobility in the Serpin Inhibitory Mechanism D.A. Lawrence. Substrate Specificity of Tissue Type Plasminogen Activator E.L. Madison. Development and Reproduction: Biology of Progesterone-Induced Uterine Serpins (P.J. Hansen, W.--J. Liu). Serpins from an Insect, Manduca Sexta M.R. Kanost, H. Jiang. Inflammation: Serpins and Programmed Cell Death G.S. Salvesen. Noninhibitor Serpins: Structure--Function Studies on PEDF: A Noninhibitory Serpin with Neurotropic Activity S.P Becerra. Abstracts: Coagulation, Neurobiology and Cancer. Fibrinolysis, Development and Reproduction. Inflammation and Noninhibitor Serpins. 10 Additional Articles. Index.

Role of Thrombin Anion-binding Exosite-I in the Formation of Thrombin-Serpin Complexes

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (k on) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77 a Q, K81Q, K109Q, K110Q, and K149 e Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereask on values were decreased 2–3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal k on values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77 a Q, where k on values were decreased 2–4-fold, compared with rIIa. For HCII/heparin, k onvalues for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149 e Q) were 2–3-fold lower than rIIa. Larger decreases in k on values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149 e Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had k onvalues reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3–7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the “hirudin-like” domain of HCII with exosite-I of thrombin.