Background: Hemostasis requires a balance between pro- and anti-coagulant factors. Hemophiliacs bleed due to a procoagulant deficiency. The 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 co-factor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of factor (F)Xa at phospholipid surfaces. Objectives: Evaluate the effects of 1) PZ or ZPI gene-deletion in hemophilia mice and 2) blocking PZ in human hemophilic plasma. Methods: 1) A Tail Vein Re-Bleeding assay (TVRB) was developed based on the serial disruption of clots forming over 15 minutes following a tail vein laceration in an anesthetized mouse. Wild type (WT)/FVIIIKO, PZKO/FVIIIKO and ZPIKO/FVIIIKO mice were evaluated in this model and their plasmas tested in thrombin generation assays. 2) A monoclonal antibody (Mab) against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results: 1) Clot formations (mean ± SEM) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice replaced 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 FVIIIKO plasma replaced with ~15% rFVIII, 2) A Mab against PZ added to human hemophilia 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.

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.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

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