Alan E. Mast

Iron deficiency in blood donors: analysis of enrollment data from the REDS-II Donor Iron Status Evaluation (RISE) study

BACKGROUND: Regular blood donors are at risk of iron deficiency, but characteristics that predispose to this condition are poorly defined.

Profiling constitutive proteolytic events in vivo

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

Iron deficiency in blood donors: the REDS‐II Donor Iron Status Evaluation (RISE) study

BACKGROUND: Blood donors are at risk of iron deficiency. We evaluated the effects of blood donation intensity on iron and hemoglobin (Hb) in a prospective study.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation

Significance The generation of thrombin by prothrombinase, a complex composed of activated (a) factors X (FXa) and V (FVa), is a final step in blood coagulation. We demonstrate that tissue factor pathway inhibitor (TFPI) blocks thrombin generation by prothrombinase at physiologically relevant rates and concentrations, but only during the initiation of clot formation. TFPI mediates this inhibitory activity through two high-affinity interactions, one with FXa and one with FVa. This is the first description of an endogenous human protein that inhibits prothrombinase under physiological conditions and may prevent a full thrombotic response to subthreshold coagulant stimuli that otherwise could occlude blood vessels. It provides unique understanding of thrombotic disorders and has important implications for development of anti-TFPI agents to treat hemophilia. Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8

Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser(174) within the connecting region between the second and third Kunitz domains ( k (cat)/ K (m) approximately 75 M(-1).s(-1)) as well as following Lys(20) within the NH(2)-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. (125)I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, (125)I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser(174), which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

Summary.  Background: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)‐anchored co‐receptor. Objectives/methods: Cellular trafficking of TFPI within aerolysin‐resistant ECV304 and EA.hy926 cells, which do not express GPI‐anchored proteins on their surface, was compared with their wild‐type counterparts. Results and conclusions: Although aerolysin‐resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild‐type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin‐resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin‐resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI‐anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co‐receptor in the ER/Golgi. The co‐receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild‐type cells or to lysosomes of aerolysin‐resistant cells. TFPI that escapes co‐receptor binding is secreted through the same pathway in both wild‐type and aerolysin‐resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Behavioral, biochemical, and genetic analysis of iron metabolism in high‐intensity blood donors

BACKGROUND: Individuals donating whole blood 13 times in a 2‐year period without development of iron deficiency anemia (superdonors) are a self‐selected population that is deferred for low hematocrit (Hct) level less frequently than other donors.

Demographic correlates of low hemoglobin deferral among prospective whole blood donors

BACKGROUND: Approximately 10% of attempted blood donations are not allowed because of low hemoglobin (Hb) deferral.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8−/−) did not rescue the embryonic death of TFPI null (Tfpi−/−) mice. Tfpi+/− did not alter the bleeding phenotype of F8−/− mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8−/− mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi+/−;F8−/− mice with hematopoietic Tfpi−/− cells compared with Tfpi+/−;F8−/− mice with Tfpi+/+ hematopoietic cells. Additionally, following femoral vein injury, Tfpi+/−;F8−/− mice with Tfpi−/− hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi+/+ hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

Characterization of the Association of Tissue Factor Pathway Inhibitor With Human Placenta

Objective—Tissue factor pathway inhibitor (TFPI) is an endothelial-associated inhibitor of blood coagulation. Because the mechanism for attachment of TFPI to endothelium is not clear, we investigated its association with human placenta. Methods and Results—Western blots demonstrate that treatment with phosphatidylinositol-specific phospholipase C (PIPLC) removes more placental TFPI than either PBS or heparin, a finding confirmed by immunohistochemistry. The amounts of heparin-releasable and PIPLC-releasable TFPI activity on placental endothelium were measured in placentas from 5 individuals. PIPLC removes >10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and >100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by ≈3-fold. An antibody specific for the C-terminal region of TFPI recognizes PIPLC-releasable TFPI in Western blots. Conclusions—GPI-anchored TFPI is the predominant form on placental endothelium. Heparin-releasable TFPI likely represents only a small portion of the total TFPI on endothelium that remains attached to cell-surface glycosaminoglycans after cleavage of the GPI anchor by endogenous enzymes. The predominance of GPI-anchored TFPI suggests that heparin infusion does not significantly redistribute TFPI within the vasculature. The intact C-terminus in GPI-anchored TFPI indicates it is not directly attached to a GPI anchor. Rather, it most likely associates with endothelium by binding to a GPI-anchored protein.