The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

BACKGROUND: ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. METHODS AND RESULTS: Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. CONCLUSION: ERp57 regulates thrombosis via multiple targets.

Vicinal thiols are required for activation of the αIIbß3 platelet integrin.

BACKGROUND: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both αIIb and ß3 of the αIIbß3 fibrinogen receptor were required for platelet aggregation. METHODS AND RESULTS: Using p-chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbß3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both αIIb and ß3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3-Dimercapto-1-propanesulfonic acid (DMPS). In contrast, the monothiol ß-mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in αIIbß3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of αIIbß3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non-labeled αIIbß3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. CONCLUSION: A vicinal thiol-containing population of αIIbß3 provides redox sensitive sites for regulation of αIIbß3.

Redox modification of platelet glycoproteins.

Platelets contain several glycoprotein receptors including the adhesion receptor glycoprotein Ib and the fibrinogen receptor glycoprotein IIbIIIa, also know as the alphaIIb betaIIIa integrin. Both of these receptors contain thiol groups and vicinal thiols representing redox sensitive sites are present in alphaIIb betaIIIa. Disulfide isomerases such as protein disulfide isomerase (PDI) that are on or recruited to the platelet surface have a role in platelet aggregation. Dynamic rearrangement of disulfide bonds in receptor signaling and platelet activation is a developing concept that requires an attacking thiol. Biochemically, a role for disulfide isomerization is suggested as the alphaIIb betaIIIa integrin undergoes major structural changes upon activation centered around a disulfide knot in the integrin. Additionally, the P2Y12 ADP receptor is involved in platelet activation by most platelet agonists and contains extracellular thiols, making it a possible site for redox modification of platelet aggregation. Various forms of redox modulation of thiols or disulfides in platelet glycoproteins exist. These include modification by low molecular weight thiols such as reduced glutathione or homocysteine, oxidized glutathione or by nitric oxide (NO) derived from s-nitrosothiols. Levels of these redox compounds change in various disease states and in some cases physiologic concentrations of these compounds have been shown to modify platelet responsiveness. Additionally, platelets themselves contain a transplasma membrane redox system capable of reducing extracellular disulfide bonds. It is likely that a redox homeostasis exists in blood with the redox environment being controlled in a way analogous to the control of ionized calcium levels or the pH of blood. Changes in this homeostasis induced by disease states or pharmacologic agents that modify the platelet redox environment will modify platelet function.

Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation.

The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin alphaIIbbeta3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and alphaIIbbeta3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as alphaIIb and beta3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of alphaIIbbeta3. In distinction to most literature reports, at least a subpopulation of alphaIIbbeta3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.

Hyperviscosity syndrome secondary to a myeloma-associated IgG(1)kappa paraprotein strongly reactive against the HIV-1 p24 gag antigen.

Hyperviscosity syndrome secondary to hypergammaglobulinemia is a rare and potentially fatal complication in patients with human immunodeficiency virus type-1 (HIV-1) infection. We studied an HIV-1-positive patient with symptomatic hyperviscosity attributable to IgG(1)kappa multiple myeloma. The patient initially responded to plasmapheresis and was subsequently treated with cytotoxic immunosuppressive chemotherapy. The patient remained asymptomatic during a 3-year follow-up period. The monoclonal IgG(1)kappa gammopathy evolved to a biclonal variant of the same subtype with an expansion of marrow plasma cell population. Western blot analysis demonstrated that this myeloma-associated paraprotein was strongly reactive against the HIV-1 p24 gag antigen.

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin.

In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.

Protein disulphide isomerase mediates platelet aggregation and secretion.

Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti-PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti-PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC-1), the rabbit anti-PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III.

The recent demonstration of a protein disulfide isomerase (PDI) on the surface of and secreted from blood platelets raises the possibility that proteins involved in hemostasis and wound healing are also substrates of this enzyme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), alpha-thrombin, and antithrombin III (AT) were used to demonstrate that PDI catalyzes formation of a TSP-thrombin-AT complex consistent with previous results with supernatant platelet activation. Concentrations of 1.25 microg/ml of PDI were sufficient to convert almost 50% of thrombin to TSP-thrombin-AT complex. Complex formation requires low concentrations of a reduced thiol and the reaction can be prevented by N-ethymaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2+ and the addition of EDTA increased the rate of complex formation, indicating that TSP in the Ca2+-free form is most effective. In the absence of AT a small amount of TSP-thrombin complex formed which was only 0-13% of maximal complex formation in the presence of AT. This result, in combination with kinetic studies showing rapid formation of thrombin-AT complex followed by conversion to ternary complex, suggests that the thrombin-AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 70% of the thrombin is incorporated into the complex in 60 min. Heparin accelerated the reaction largely by enhancing formation of thrombin-AT complexes and had little effect on TSP. PDI coprecipitated with TSP from the supernatant solution of activated platelets, suggesting an association between PDI and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other proteins.

A polyclonal antibody to protein disulfide isomerase induces platelet aggregation and secretion.

Monoclonal mouse antiplatelet antibodies against a variety of platelet surface components can activate platelets, causing platelet aggregation and secretion. The mechanism involves binding of the Fab domain to a platelet surface antigen, and the activation occurs through an interaction of the Fc domain with the platelet FcgammaRII receptor. There is almost no information on FcgammaRII receptor-dependent activation of platelets by polyclonal rabbit antibodies. We presently report that a polyclonal rabbit antibody to a platelet surface antigen, protein disulfide isomerase, induces platelet aggregation and secretion. These effects are seen with concentrations of the antiprotein disulfide isomerase antibody as low as 25 to 40 microg/mL. Fab and F(ab')2 preparations of the rabbit antiprotein disulfide isomerase antibody do not cause aggregation. Fab made from the rabbit antiprotein disulfide isomerase antibody as well as a monoclonal antibody to the FcgammaRII (IV.3) receptor block the aggregation and secretion responses. Aggregation and secretion are inhibited by an antiglycoprotein IIbIIIa antibody, which blocks fibrinogen binding and wortmannin, an inhibitor of phosphoinositide 3-kinase. Aspirin, prostaglandin E1, and Ethylenediamine-tetraacetic acid (EDTA) also block the platelet responses. These data suggest that activation of platelets by polyclonal antibodies occurs by mechanisms similar to those found with activating monoclonal antibodies.

Measurement of soluble transferrin receptor in serum of healthy adults.

The concentration of soluble transferrin receptor (sTfR) in serum is reported to be useful in the diagnosis of iron deficiency, especially for patients with concurrent chronic disease, where routine tests of iron status are compromised by the inflammatory condition. A new diagnostic assay for sTfR is calibrated against natural plasma sTfR, thus minimizing calibration discrepancies that result from differences between the analyte and the cellular transferrin receptor used in other assays. Use of the new assay to measure sTfR concentrations in 225 healthy, hematologically normal adults provided a reference interval against which pathological samples could be compared. There was no difference in the reference intervals for men and women and no correlation of [sTfR] with the age of the subject. Black subjects had significantly higher concentrations than nonblacks, and people living at high altitude had higher concentrations than those living closer to sea level. These differences were additive.

Late-onset warfarin-induced skin necrosis: case report and review of the literature.

Warfarin-induced skin necrosis is a rare complication of therapy with warfarin or other coumarin derivatives. When it occurs it usually appears 3 to 6 days after initiation of therapy and almost always between days 1 and 10. We report a case of late-onset (16 days after initiation of therapy) warfarin-induced skin necrosis and review the literature on this rarely reported variant of warfarin-induced skin necrosis. The skin lesion in our patient was not associated with either deficiency of protein C or resistance to activated protein C.

Human endothelial cells in culture and in vivo express on their surface all four components of the glycoprotein Ib/IX/V complex.

The platelet glycoprotein Ib (GpIb) complex is composed of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta and the noncovalently associated GpIX and GpV. GpIb alpha contains binding sites for von Willebrand factor and for thrombin and mediates platelet adhesion to the subendothelium under conditions of high shear stress. We have previously shown the presence of GpIb alpha and GpIb beta mRNA and protein in cultured human umbilical vein endothelial cells (HUVECs) as well as the presence of GpIb alpha mRNA and protein in tonsillar endothelium. We, therefore, probed ECs for the presence of the other components of the GpIb/IX/V complex. We have identified the presence of GpIX and GpV mRNA in cultured HUVEC monolayers. The sequence of HUVEC GpIX cDNA was identical to the previously published human erythroleukemia (HEL) cell GpIX cDNA sequence. Two species of GpV mRNA, one of 3 kb and one of 4.4 kb, were found in HUVECs, whereas HEL cells displayed only the 4.4-kb species and the megakaryocytic cell line CHRF-288 contained only the 3-kb species. We previously showed that EC GpIb alpha protein is identical in molecular weight to platelet GpIb alpha. HUVEC GpIb beta, in contrast to its platelet counterpart, has a molecular weight of 50 kD and forms a correspondingly larger disulfide-bonded complex with EC GpIb alpha. The molecular weights of GpIX and GpV were 22 and 88 kD, respectively, identical to the corresponding platelet polypeptides. Furthermore, we have identified all four components of the complex in tonsillar vessels. Using flow cytometry, we have established that all four polypeptides of the GpIb/IX/V complex are expressed on the surface membranes of cultured HUVECs and adult aortic ECs. Furthermore, using two-color fluorescence, we have shown that all ECs expressing GpIb alpha also express GpIX and GpV on their surface. The ratio of GpIb alpha:GpIX:GpV is 1:1:0.5, which is identical to the ratio present in platelets. None of the polypeptides of the GpIb complex could be identified on the surface of human smooth muscle cells or lymphocytes. The presence of all members of the GpIb complex in the EC membrane suggests that this complex may play a role in endothelial function in vivo.

Lactic acidosis secondary to severe anemia in a patient with paroxysmal nocturnal hemoglobinuria.

A patient with paroxysmal nocturnal hemoglobinuria developed lactic acidosis associated with severe anemia. The lactic acidosis corrected after blood transfusion. In the absence of shock, sepsis, or other identifiable causes of lactic acidosis, the severe anemia (hemoglobin 1.2 g/dl) appeared to be the primary etiologic factor.

Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase.

Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [3H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys974 was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37 degrees C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.

Warfarin-induced skin necrosis occurring four days after discontinuation of warfarin.

Warfarin-induced skin necrosis is a rare complication of warfarin therapy. It appears between days 1 and 10 of therapy while the patient is receiving warfarin. Warfarin-induced skin necrosis occurring several days after cessation of therapy has not been reported. We report an atypical case of warfarin-induced skin necrosis which occurred 96 h after the last dose of warfarin was given while the prothrombin time was still elevated.

Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes the formation as well as the isomerization of disulfide bonds. In this study, antibodies against PDI were used to show PDI antigen on the platelet surface by indirect immunofluorescence microscopy and by flow cytometry. The platelets were not activated, as evidenced by the absence of staining by an antibody against P-selectin. Permeabilized platelets showed little cytosolic PDI by indirect immunofluorescence microscopy, suggesting that the majority of platelet PDI is localized to the platelet surface. PDI activity against "scrambled" RNase was shown with intact platelets. The activity was inhibited by inhibitors of PDI and by an antibody against PDI. Other blood cells showed little PDI. Platelet surface PDI may play a role in the various physiological and pathophysiologic processes in which platelets are involved.

Characterization of protein disulphide isomerase released from activated platelets.

Protein disulphide isomerase (PDI) activity is released by activated platelets. In this study, PDI was purified from platelets and found to have an apparent mass, pI and N-terminal sequence similar to those for other human PDIs. Rabbit antibodies were generated and used to establish that, on activation, platelets release a protein immunologically identical to PDI in platelets. Approximately 10% of total platelet PDI was released by thrombin and 20% by calcium ionophore. The antibody was used to demonstrate PDI on the external surface of platelets by electron microscopy. Flow cytometry was used to demonstrate that upon activation of platelets with ionophore PDI was released by vesiculation. Since platelets are present and become activated at sites of vascular injury, platelet PDI may play a role in the various haemostatic and tissue remodelling processes in which platelets are involved.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.