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Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood "macrophage disappearance reaction." Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity., (© 2019 Zhang et al.)

Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIX WT ) in null (cross-reactive material negative, CRM - ) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 is several-fold the FIX level measured in plasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIX WT and the increased half-life FIXs Alprolix (FIX FC ) and Idelvion (FIX Alb ) produce comparable hemostatic results in CRM - mice. In bleeding CRM - hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIX WT < FIX FC < FIX Alb Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM + ). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM + mice expressing a dysfunctional FIX, FIX R333Q , that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The results in this mouse model of CRM + hemophilia B demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy., (© 2019 by The American Society of Hematology.)

Essentials Protein Z (PZ) catalyzes PZ-dependent proteinase inhibitor (ZPI) inactivation of factor (F)Xa. Gene-deletion of PZ or ZPI improves coagulation in hemophilia (FVIII knockout) mice. A PZ blocking antibody enhances thrombin generation in human hemophilia plasma. Suppression of the PZ/ZPI pathway may ameliorate the phenotype of severe hemophilia. SUMMARY: Background Hemostasis requires a balance between procoagulant and anticoagulant factors. Hemophiliacs bleed because of a procoagulant deficiency. Targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a cofactor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of activated factor X at phospholipid surfaces. Objectives To evaluate the effects of PZ or ZPI gene deletion in hemophilic mice, and of blocking PZ in human hemophilic plasma. Methods A tail vein rebleeding assay (TVRB) was developed on the basis of the serial disruption of clots forming over a period of 15 min following tail vein laceration in an anesthetized mouse. Wild-type (WT)/FVIII knockout FVIIIKO, PZ knockout PZKO/FVIIIKO and ZPI knockout ZPIKO/FVIIIKO mice were evaluated in this model, and their plasmas were tested in thrombin generation assays. A mAb against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results The numbers of clot disruptions (mean ± standard error of the mean) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice supplemented with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to that in FVIIIKO plasma supplemented with ~ 15% recombinant FVIII. A mAb against PZ added to human hemophilic plasma enhanced thrombin generation to an extent similar to the addition of ~ 15% FVIII. Conclusions Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia., (© 2018 International Society on Thrombosis and Haemostasis.)

Essentials Mouse models are often used to define roles of tissue factor pathway inhibitor (TFPI) in man. TFPI isoform-specific KOs reveal unexpected differences between mouse and human TFPI physiology. Mouse plasma contains 20 times more TFPI than man, derived from TFPIγ, a form not found in man. TFPIγ null mice, expressing only TFPI isoforms α and β, may better reflect the human situation. SUMMARY: Background Mouse models can provide insight into the pathophysiology of human thrombosis and hemostasis. Tissue factor pathway inhibitor (TFPI) regulates coagulation through protein S (PS)-enhanced factor (F) Xa inhibition and FXa-dependent inhibition of FVIIa/tissue factor (TF) activity. TFPI is expressed as isoforms α and β in man, and α, β and γ in the mouse. Objective Assess the reliability of extending TFPI-related studies in mice to humans. Method Compare mouse and human TFPI physiology using a variety of methods. Results Mouse TFPI and human TFPI are similar in regard to: (i) the mechanisms for FVIIa/TF and FXa inhibition; (ii) TFPIα is a soluble form and TFPIβ is glycosyl phosphatidyl inositol (GPI) membrane anchored; (iii) the predominant circulating form of TFPI in plasma is lipoprotein-associated; (iv) low levels of TFPIα circulate in plasma and increase following heparin treatment; and (v) TFPIα is the isoform in platelets. They differ in that: (i) mouse TFPI circulates at a ~20-fold higher concentration; (ii) mouse lines with isolated isoform deletions show this circulating mouse TFPI is derived from TFPIγ; (iii) sequences homologous to the mouse TFPIγ exon are present in many species, including man, but in primates are unfavorable for splicing; and (iv) tandem mass spectrometry (MS/MS) detects sequences for TFPI isoforms α and β in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIβ, studies in TFPIγ null mice, expressing only α and β, only α or only β should better reflect the human situation., (© 2018 International Society on Thrombosis and Haemostasis.)

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

Acquired factor X (FX) deficiency unrelated to amyloidosis is a rare disorder in which an anti-FX antibody is infrequently detected. A patient with severe bleeding due to a calcium ion-dependent anti-FX IgG antibody is described. The FX affinity purified IgG bound the light chain of FX, but not FX lacking its γ-carboxyglutamic acid domain, and binding was enhanced >1000-fold in the presence of calcium ions. The antibody also recognized prothrombin and factor VII with about 100-fold and 1000-fold lower affinity. Like a lupus anticoagulant, increasing concentrations of phospholipids in functional assays reduced the inhibitory activity of the antibody. The effect of these properties of the inhibitor on laboratory diagnostic studies is considered., (© 2013.)

Objective: Sepsis, a leading cause of mortality in critically ill patients, is closely linked to the excessive activation of coagulation and inflammation. Protein Z, a cofactor for the protein Z-dependent protease inhibitor, enhances the inhibition of coagulation factor Xa, and protein Z-dependent protease inhibitor inhibits factor XIa in a protein Z-independent fashion. The functions of protein Z and protein Z-dependent protease inhibitor in the inflammatory and coagulant responses to septic illness have not been evaluated., Design: For induction of generalized Shwartzman reaction, dorsal skinfold chamber-equipped mice were challenged twice with lipopolysaccharide (0.05 mg/kg on day -1 and 5 mg/kg body weight 24 hr later). Time-matched control animals received equal volumes of saline., Setting: University research laboratory., Subjects, Interventions, and Measurements: Using intravital fluorescence microscopy in protein Z-dependent protease inhibitor deficient (ZPI) and protein Z deficient (PZ) mice, as well as their wild-type littermates (ZPI, PZ), kinetics of light/dye-induced thrombus formation and microhemodynamics were assessed in randomly chosen venules. Plasma concentrations of chemokine (C-X-C motif) ligand 1, interleukin-6, and interleukin-10 were measured. Liver and lung were harvested for quantitative analysis of leukocytic tissue infiltration and thrombus formation., Main Results: After induction of generalized Shwartzman reaction, all mice showed significant impairment of microhemodynamics, including blood flow velocity, volumetric blood flow, and functional capillary density, as well as leukocytopenia and thrombocytopenia. Thrombus formation time was markedly prolonged after induction of generalized Shwartzman reaction in all mice, except of ZPI mice, which also had a significantly higher fraction of occluded vessels in liver sections. PZ mice developed the highest concentrations of interleukin-6 and interleukin-10 in response to generalized Shwartzman reaction and showed greater leukocytic tissue infiltration than their wild-type littermates., Conclusions: In this murine model of generalized Shwartzman reaction, protein Z-dependent protease inhibitor deficiency enhanced the thrombotic response to vascular injury, whereas protein Z deficiency increased inflammatory response.

In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis.

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.

Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

TFPI is a multivalent, Kunitz-type proteinase inhibitor, which, due to alternative mRNA splicing, is transcribed in three isoforms: TFPIalpha, TFPIdelta, and glycosyl phosphatidyl inositol (GPI)-anchored TFPIbeta. The microvascular endothelium is thought to be the principal source of TFPI and TFPIalpha is the predominant isoform expressed in humans. TFPIalpha, apparently attached to the surface of the endothelium in an indirect GPI-anchor-dependent fashion, represents the greatest in vivo reservoir of TFPI. The Kunitz-2 domain of TFPI is responsible for factor Xa inhibition and the Kunitz-1 domain is responsible for factor Xa-dependent inhibition of the factor VIIa/tissue factor catalytic complex. The anticoagulant activity of TFPI in one-stage coagulation assays is due mainly to its inhibition of factor Xa through a process that is enhanced by protein S and dependent upon the Kunitz-3 and carboxyterminal domains of full-length TFPIalpha. Carboxyterminal truncated forms of TFPI as well as TFPIalpha in plasma, however, inhibit factor VIIa/tissue factor in two-stage assay systems. Studies in gene-disrupted mice demonstrate the physiological importance of TFPI.

Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner. The observed 50% inhibitory concentration (IC(50)) for the HRPII neutralization of AT activity is approximately 30nM for factor Xa inhibition and 90nM for thrombin inhibition. Zn(2+) was required for these reactions with a distribution coefficient (K(d)) of approximately 7μM. Substituting Zn(2+) with Cu(2+), but not with Ca(2+), Mg(2+), or Fe(2+), maintained the HRPII effect. HRPII attenuated the prolongation in plasma clotting time induced by heparin, suggesting that HRPII inhibits AT activity by preventing its stimulation by heparin. In the microvasculature, where erythrocytes infected with P falciparum are sequestered, high levels of released HRPII may bind cellular glycosaminoglycans, prevent their interaction with AT, and thereby contribute to the procoagulant state associated with P falciparum infection.

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.

Protein S (PS) enhances the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-alpha (TFPI-alpha) in the presence of Ca(2+) and phospholipids. Altered forms of recombinant TFPI-alpha were used to determine the structures within TFPI-alpha that may be involved in this PS-dependent effect. Wild-type TFPI-alpha (TFPI(WT)), TFPI-alpha lacking the K3 domain (TFPI-(DeltaK3)), and TFPI-alpha containing a single amino acid change at the putative P1 residue of K3 (R199L, TFPI(K3P1)) produced equivalent FXa inhibition in the absence of PS, whereas the response in FXa inhibition produced by PS was reduced with TFPI(K3P1) (EC(50) 61.8 +/- 13.4nM vs 8.0 +/- 0.4nM for TFPI(WT)) and not detectable with TFPI-(DeltaK3). Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPI(WT) and TFPI-(DeltaK3) but not the isolated K3 domain, whereas PS bound TFPI(WT) and the K3 domain but not TFPI-(DeltaK3). Addition of TFPI(WT), TFPI(K3P1), or TFPI-(DeltaK3) produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPI(WT) was substantially greater than that of TFPI(K3P1) > TFPI-(DeltaK3) in normal plasma and PS-deficient plasma reconstituted with PS. We conclude that the PS-mediated enhancement of FXa inhibition by TFPI-alpha involves an interaction between PS and TFPI-alpha, which requires the K3 domain of TFPI-alpha.

Summary Introduction: Protein Z is a vitamin K-dependent protein that serves as a cofactor for the inhibition of activated factor X by the serpin protein Z-dependent protease inhibitor (ZPI). Protein Z plasma levels have been shown to be reduced in patients with peripheral arterial disease (PAD), but ZPI levels have not yet been reported. The aim of this study was to more fully assess the protein Z-ZPI system in individuals with atherosclerosis selected by the presence of symptomatic PAD., Materials and Methods: Protein Z and ZPI levels were determined in 95 PAD patients (73 males; 22 females) [median age: 73 years (range, 50-86 years)] and in 190 controls comparable for age and gender. Protein Z was measured using a commercial immunoassay, and ZPI was measured with a homemade immunoassay and a functional assay., Results: Protein Z antigen, ZPI antigen and ZPI function were found to be significantly lower in PAD patients with respect to controls [protein Z, median 72.5% (range: 3.4-123.7%) vs. 90.7% (range: 32.1-203.2%), P < 0.0001; ZPI antigen, 86.1% (range: 25.1-149.5%) vs. 93.2% (range: 48.9-171.3%), P = 0.004; ZPI function, 83.5% (range: 21.1-135.2%) vs. 97.2% (range: 50.5-175.5%), P < 0.0001]. The lowest tertiles of protein Z antigen [odds ratio (OR) 5.4, 95% confidence interval (CI) 2.2-13.5, P < 0.0001] and ZPI function (OR 2.4, 95% CI 1.1-5.5, P = 0.03) were associated with PAD on multivariate analysis after adjustment for age, gender, and traditional cardiovascular risk factors. A significant inverse relationship was also observed between protein Z and ZPI levels and the number of traditional cardiovascular risk factors and the clinical severity of disease (Fontaine stage)., Conclusions: Low levels of protein Z antigen and protein Z activity are significantly associated with the occurrence and severity of atherosclerotic PAD.

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

Protein Z (PZ) is a plasma vitamin K-dependent protein that functions as a cofactor to dramatically enhance the inhibition of coagulation factor Xa by the serpin, protein Z-dependent protease inhibitor (ZPI). In vitro, ZPI not only inhibits factor Xa in a calcium ion-, phospholipid-, and PZ-dependent fashion, but also directly inhibits coagulation factor XIa. In murine gene-deletion models, PZ and ZPI deficiency enhances thrombosis following arterial injury and increases mortality from pulmonary thromboembolism following collagen/epinephrine infusion. On a factor V(Leiden) genetic background, ZPI deficiency produces a significantly more severe phenotype than PZ deficiency, implying that factor XIa inhibition by ZPI is physiologically relevant. The studies in mice suggest that human PZ and ZPI deficiency would be associated with a modest thrombotic risk with ZPI deficiency producing a more severe phenotype.

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Decreased fibrinolytic function favors the development of pulmonary fibrosis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a strong suppressor of fibrinolysis, but its role in lung fibrosis is unknown. Therefore, we compared bleomycin-induced lung fibrosis in TAFI-deficient, heterozygous, and wild-type mice. The animals were sacrificed 21 days after bleomycin administration, and markers of lung fibrosis and inflammation were measured. The bronchoalveolar lavage fluid levels of total protein, neutrophil proteases (elastase, myeloperoxidase), cytokines (tumor necrosis factor-alpha, interleukin-13), chemokine (monocyte chemoattractant protein-1), coagulation activation marker (thrombin-antithrombin complex), total soluble collagen, and growth factors (platelet-derived growth factor, transforming growth factor-beta1, granulocytic-macrophage growth factor) were significantly decreased in knockout mice compared to wild-type mice. Further, histological findings of fibrosis, fibrin deposition, and hydroxyproline and collagen content in the lung were significantly decreased in knockout mice compared to wild-type mice. Depletion of fibrinogen by ancrod treatment led to equalization in the amount of fibrosis and collagen deposition in the lungs of knockout and wild-type mice. No difference was detected in body temperature or arterial pressure between the different mouse phenotypes. These results suggest that the anti-fibrinolytic activity of TAFI promotes lung fibrosis by hindering the rate at which fibrin is degraded.

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, severe von Willebrand disease, and other rare factor deficiencies. A novel approach to improve coagulation in such clotting disorders has been identified that, parodoxically, involves heparinlike sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). A mechanism involving blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI) by NASPs, was conceived as an approach for improving procoagulant behavior in hemophilic settings. A subset of NASPs, including pentosan polysulfate (PPS) and fucoidan inhibited both full-length and Kunitz 1 and 2 (K1K2) TFPI and, at concentrations from 4-500 nM, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced factor VII levels when tested in dilute prothrombin time (dPT) assays. Fucoidan did not reduce normal plasma APTT times implying specificity for extrinsic pathway control. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Increased survival of factor deficient mice following a bleeding challenge was observed. Accordingly, administration of select NASP(s), via mechanism(s) not fully understood, represents a unique means of improving coagulation in bleeding disorders.

Background: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage., Objective: Compare the structure and properties of TFPIalpha and TFPIbeta., Methods: TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF)., Results and Conclusions: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.

To assess the potential roles of protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) in venous thrombosis, their plasma levels were measured in 426 individuals with venous thrombosis and 471 control individuals participating in the Leiden Thrombophilia Study. A relationship between the level of PZ or ZPI and venous thrombosis was not detected in the overall case-control study. PZ and ZPI circulate as a complex and their plasma levels are interdependent. Both PZ and ZPI are increased with oral contraceptive use and reduced with oral anticoagulant therapy.

Background: Tissue factor pathway inhibitor (TFPI)-alpha, a key regulator of tissue factor-induced coagulation, contains 3 tandem Kunitz-type inhibitory domains. Kunitz-1 binds and inhibits factor VIIa in the factor VIIa/tissue factor complex, and Kunitz-2 binds and inhibits factor Xa. The role of the Kunitz-3 domain of TFPI-alpha, however, has remained an enigma., Methods and Results: To determine the structures within TFPI-alpha involved in its binding to cell surface, altered forms of TFPI-alpha were expressed in C127 (mouse mammary) cells: C-terminal truncated forms TFPI-alpha (252), TFPI-alpha (242), and TFPI-alpha (181), which also lacks the third Kunitz domain (K3); TFPI-alpha (desK3), which lacks only the K3 domain; and TFPI-alpha (R199L), in which the putative P1 site in K3 is changed from arginine to leucine. By flow cytometry (fluorescence-activated cell sorting), the altered forms 252, 242, and R199L showed significantly reduced binding, whereas the forms 181 and desK3 completely failed to bind to the cell surface. Transient expression of WT-, desK3-, and K3/K2-TFPI-alpha (in which K3 is replaced with K2) in another cell line (b-end3, mouse endothelial) produced comparable results. Exogenously added C-terminal truncated and R199L forms of TFPI-alpha bound poorly and desK3 did not bind at all to the surface of ECV304 cells in which TFPI-alpha expression had been "knocked down" by RNA interference., Conclusions: Optimal cell binding of endogenously expressed TFPI-alpha requires its K3 and C-terminal domains, and within the K3 domain, the P1 (R199) residue plays an important role. Thus, one role of the K3 domain involves the cell surface localization of TFPI-alpha.