Suppressing protein Z-dependent inhibition of factor Xa improves coagulation in hemophilia A.

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.

Re-evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology.

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 ß, 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 ß in man, and α, ß 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 TFPIß 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 ß in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIß, studies in TFPIγ null mice, expressing only α and ß, only α or only ß should better reflect the human situation.

Effect of tissue factor deficiency on mouse and tumor development.

Previous reports suggest that tissue factor (TF) may play an essential role in embryonic vascular development and tumor angiogenesis. To further examine this relationship, the morphology of fully developed TF-deficient embryos and the growth of TF-deficient teratomas and teratocarcinomas were analyzed. In a 129/Sv genetic background, TF null embryos do not survive beyond mid-gestation. In contrast, 14% of 129/Sv x C57BL/6 TF-deficient embryos escape this early mortality and survive to birth. On gross and microscopic inspection, these late gestation, TF-deficient embryos appear normal. The growth and vascularity of TF(+/+), TF(+/-), and TF(-/-) teratomas and teratocarcinomas are indistinguishable. Thus, tumor-derived TF is not required for tumor growth and angiogenesis and the combined data do not support an essential role for TF in embryonic vascular development.

A murine model of factor XI deficiency.

To facilitate investigations into the physiologic and pathologic roles of factor XI, we have developed a murine model of severe factor XI deficiency using the technique of homologous recombination in embryonic stem cells. The factor XI gene was disrupted by introducing a neomycin phosphotransferase gene into the fifth exon. The activated partial thromboplastin times of homozygous null mice were prolonged (158- > 200 s) compared with wild type (25-34 s) and heterozygous null (40-61 s) litter mates. Factor XI activity was absent from the plasma of mice homozygous for the null mutation and factor XI mRNA was undetectable by Northern blot and reverse transcription/PCR in the livers of homozygous null animals. The genotypes of progeny from matings of mice heterozygous for the factor XI null allele followed the expected Mendelian ratio (1:2:1, wild type 26%, heterozygote null 54%, homozygous null 20%), indicating that severe factor XI deficiency did not result in increased intrauterine death. Results of a tail transection bleeding time assay were similar for wild type and homozygous null animals with, at most, a tendency for slightly prolonged bleeding in the homozygous null animals. The factor XI deficient mice are a unique tool for evaluating the role of factor XI in normal hemostasis and pathologic coagulation.

Targeted disruption of the murine tissue factor gene results in embryonic lethality.

Tissue factor (TF) is an integral membrane glycoprotein that is believed to be the physiologic initiator of the blood coagulation cascade. Disruption of the mouse tissue factor gene leads to embryonic lethality between days E9.5-E11.5 of gestation. On E9.5, TF(-/-) embryos appear indistinguishable from their TF(+/+) and TF(+/-) littermates. By E10.5, TF(-/-) embryos are severely growth retarded, appear nearly bloodless, and are in most cases dead. Initial observations suggest that TF(-/-) embryos are dying of circulatory failure. Approximately 15% of the TF(-/-) embryos survive beyond E10.5, but none complete gestation. Heterozygotes appear normal and free of bleeding complications.