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There have been substantial advances in vascular protein disulfide isomerases (PDIs) in platelet function and thrombosis in recent years. There are 4 known prothrombotic thiol isomerases; PDI, endoplasmic reticulum protein (ERp)57, ERp72, and ERp46, and 1 antithrombotic PDI; transmembrane protein 1. A sixth PDI, ERp5, may exhibit either prothrombotic or antithrombotic properties in platelets. Studies on ERp46 in platelet function and thrombosis provide insight into the mechanisms by which these enzymes function. ERp46-catalyzed disulfide cleavage in the α IIb β 3 platelet integrin occurs prior to PDI-catalyzed events to maximally support platelet aggregation. The transmembrane PDI transmembrane protein 1 counterbalances the effect of ERp46 by inhibiting activation of α IIb β 3 . Recent work on the prototypic PDI found that oxidized PDI supports platelet aggregation. The a' domain of PDI is constitutively oxidized, possibly by endoplasmic reticulum oxidoreductase-1α. However, the a domain is normally reduced but becomes oxidized under conditions of oxidative stress. In contrast to the role of oxidized PDI in platelet function, reduced PDI downregulates activation of the neutrophil integrin αMβ 2 . Intracellular platelet PDI cooperates with Nox1 and contributes to thromboxane A2 production to support platelet function. Finally, α IIb and von Willebrand factor contain free thiols, which alter the functions of these proteins, although the extent to which the PDIs regulate these functions is unclear. We are beginning to understand the substrates and functions of vascular thiol isomerases and the redox network they form that supports hemostasis and thrombosis. Moreover, the disulfide bonds these enzymes target are being defined. The clinical implications of the knowledge gained are wide-ranging., Competing Interests: Declaration of competing interests The authors declare no conflict of interest., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Five secreted platelet protein disulfide isomerases (PDIs) and 1 transmembrane PDI regulate platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first member of the PDI family found to negatively regulate platelet aggregation and platelet accumulation in vivo . The effect of TMX1 on coagulation is unknown., Objectives: To determine the effect of TMX1 on coagulation., Methods: TMX1 -/- mice were used to study platelet accumulation and fibrin deposition in vivo in the laser-induced thrombosis injury model. Annexin V deposition at the site of vascular injury was studied using conditional TMX1 knockout mice. Annexin V binding to platelets was studied using human platelets, anti-TMX1 antibodies, and TMX1-deficient platelets., Results: TMX1 -/- mice had increased fibrin deposition that was reversed with infusion of recombinant TMX1. Infusion of recombinant TMX1 inhibited platelet accumulation and fibrin deposition in wild-type mice and inhibited fibrin deposition in β 3 -null mice. Platelet accumulation is absent in β 3 -null mice, suggesting that TMX1 inhibits coagulation independently of platelets. Annexin V binding was increased in activated human platelets incubated with an anti-TMX1 antibody and mouse platelets lacking TMX1. Addition of recombinant TMX1 decreased annexin V binding to platelets. Annexin V binding was increased at the site of vascular injury in Tie2-Cre/TMX1 fl/fl mice deficient in endothelial cell TMX1., Conclusion: TMX1 decreases coagulation at the site of vascular injury and negatively regulates phosphatidylserine exposure on endothelial cells and platelets., (© 2024 The Author(s).)

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1 +/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H 2 O 2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3 and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs and in contrast to PDI, generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds., (© 2022 by The American Society of Hematology.)

There has recently been considerable progress of the field of extracellular protein disulfide isomerases with vascular thiol isomerases in the forefront. Four members of protein disulfide isomerase (PDI) family of enzymes, PDI, ERp57, ERp72, and ERp5, have been shown to be secreted from activated platelets and endothelial cells at the site of vascular injury. Each isomerase individually supports platelet accumulation and coagulation, as indicated by multiple levels of evidence, including inhibitory antibodies, targeted knockout mice, and mutant isomerases. The transmembrane PDI family member TMX1 was recently shown to inhibit platelet function and thrombosis, demonstrating that the PDIs can have opposing functions in thrombosis. These observations provide a new concept that thiol isomerases can both positively and negatively regulate hemostasis, constituting off-on redox switches controlling activation of hemostatic factors. This redox network serves to maintain vascular homeostasis. Integrins such as the αIIbβ3 fibrinogen receptor on platelets appear to be major substrates, with the platelet receptor for von Willebrand factor, glycoprotein Ibα, as another substrate. S-nitrosylation of the prothrombotic PDIs may additionally negatively regulate platelets and thrombosis. Thiol isomerases also regulate coagulation in mouse models, and a clinical trial with the oral PDI inhibitor isoquercetin substantially decreased markers of coagulation in patients at risk for thrombosis. This review updates recent findings in the field and addresses emerging evidence that thiol/disulfide-based reactions mediated by the prothrombotic secreted PDIs are balanced by the transmembrane member of this family, TMX1., (© 2020 International Society on Thrombosis and Haemostasis.)

Essentials Protein disulfide isomerase (PDI) interacts with the αIIbβ3 integrin on platelets We generated PDI domain fragments and full-length PDI containing point mutations PDI interacts with αIIbβ3 through the b' domain, with the a and a' domains contributing This is the first report demonstrating PDI binding to a native protein on intact cells SUMMARY: Background Protein disulfide isomerase (PDI) is an oxidoreductase consisting of four domains arranged in the order a-b-b'-a' with an x-linker between the b' and a' domains. PDI binds to α II b β 3 integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet α II b β 3 is unknown. Objective and methods We used PDI domain fragments and full-length PDI containing point mutations to study inhibition of Alexa 488-labeled PDI binding to thrombin-activated platelets. The effect of the PDI variants on platelet aggregation was tested. Results Only PDI fragments containing the b' domain bound to activated platelets. A double mutant of the b' domain had decreased binding, confirming the essential role of the b' domain. Addition of mutations in the a and a' domains further decreased binding, suggesting that these domains contribute to the interaction of PDI with platelets. The ability of the b' domain to interact directly with α II b β 3 was demonstrated with surface plasmon resonance, with contributions from the a and a' domains. The abb'x PDI fragment that binds to platelets but lacks the critical C-terminal active site inhibited platelet aggregation and in vivo thrombosis. Moreover, site mutations in the a, b' and a' domains that resulted in partial binding to platelets partially recovered aggregation of PDI-null platelets. PDI mutants that did not bind showed no recovery. Conclusion PDI functionally interacts with α II b β 3 on platelets through the substrate-binding b' domain, with the a and a' domains contributing to efficient binding., (© 2018 Temple University. International Society on Thrombosis and Haemostasis.)

Secreted platelet protein disulfide isomerases, PDI, ERp57, ERp5, and ERp72, have important roles as positive regulators of platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first described transmembrane member of the protein disulfide isomerase family of enzymes. Using a specific antibody, the recombinant extracellular domain of TMX1 (rTMX1) protein, a knockout mouse model, and a thiol-labeling approach, we examined the role of TMX1 in platelet function and thrombosis. Expression of TMX1 on the platelet surface increased with thrombin stimulation. The anti-TMX1 antibody increased platelet aggregation induced by convulxin and thrombin, as well as potentiated platelet ATP release. In contrast, rTMX1 inhibited platelet aggregation and ATP release. TMX1-deficient platelets had increased aggregation, ATP release, αIIbβ3 activation, and P-selectin expression, which were reversed by addition of rTMX1. TMX1-knockout mice had increased incorporation of platelets into a growing thrombus in an FeCl 3 -induced mesenteric arterial injury model, as well as shortened tail-bleeding times. rTMX1 oxidized thiols in the αIIbβ3 integrin and TMX1-deficient platelets had increased thiols in the β3 subunit of αIIbβ3, consistent with oxidase activity of rTMX1 against αIIbβ3. Thus, TMX1 is the first identified extracellular inhibitor of platelet function and the first disulfide isomerase that negatively regulates platelet function., (© 2019 by The American Society of Hematology.)

Purpose of Review: The present review provides an overview of recent findings on new members of the protein disulfide isomerase (PDI) family required for thrombosis., Recent Findings: Twenty years ago PDI was shown to mediate platelet aggregation, and 10 years ago PDI was shown to support thrombosis in vivo. Subsequently, other members of this endoplasmic reticulum family of enzymes, ERp57 and ERp5, were demonstrated to support thrombosis. A fourth member, ERp72, was recently shown to be required for platelet accumulation and fibrin deposition in vivo. None of these enzymes can individually support these processes. Moreover, aggregation of platelets deficient in a specific PDI is only recovered by the PDI that is missing. This implies that each PDI has a distinct role in activation of the αIIbβ3 fibrinogen receptor and platelet aggregation. Free thiols can be labeled in both subunits of αIIbβ3, suggesting cysteine-based reactions are involved in relaying conformational changes from the cytoplasmic tails to the integrin headpiece of this integrin., Summary: Multiple members of the PDI family support platelet function, and hemostasis and thrombosis with distinct roles in these processes. The individual cysteine targets of each enzyme and how these enzymes are integrated into a network that supports hemostasis and thrombosis remain to be elucidated.

Protein disulfide isomerase (PDI) plays an important role in fibrin generation in vivo, but the underlying mechanism remains largely unknown. In this study, using thrombin generation assay (TGA), we investigated whether PDI contributes to tissue factor (TF)-mediated thrombin generation. Human peripheral blood mononuclear cells (PBMCs) were treated with 100 ng/ml lipopolysaccharide (LPS), the expression of TF on cell surface was analyzed by flow cytometry. After incubation with an inhibitory anti-TF antibody, recombinant PDI protein or a PDI inhibitor PACMA31, LPS-stimulated human PBMCs were incubated with human plasma, and thrombin generation was assessed by Ceveron Alpha TGA and a fluorescent thrombin substrate. Bone marrow mononuclear cells isolated from PDI-knockout and wild-type mice were stimulated by LPS, followed by measurement of thrombin generation. LPS stimulation increased expression of TF on PBMCs, and thrombin generation. Inhibitory anti-TF antibody almost completely suppressed thrombin generation of LPS-stimulated PBMCs, suggesting that thrombin generation was TF-dependent. Recombinant PDI protein increased thrombin generation, while PACMA31 attenuated thrombin generation. Compared with control cells, PDI-deficient marrow mononuclear cells had less capacity in thrombin generation. Taken together, these data suggest that PDI enhances TF-dependent thrombin generation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Several CGHC motif-containing disulfide isomerases support thrombosis. We here report that endoplasmic reticulum protein 72 (ERp72), with 3 CGHC redox-active sites (a o , a, and a'), supports thrombosis. We generated a new conditional knockout mouse model and found that Tie2-Cre/ERp72 fl/fl mice with blood and endothelial cells lacking ERp72 had prolonged tail bleeding times and decreased platelet accumulation in laser-induced cremaster arteriole injury and FeCl 3 -induced mesenteric arterial injury. Fibrin deposition was decreased in the laser injury model. Both platelet and fibrin accumulation defects were fully rescued by infusion of recombinant ERp72 containing functional a and a' CGHC motifs (ERp72(oo-ss-ss)). Infusion of ERp72 containing inactivated a and a' CGHC motifs (ERp72(ss-oo-oo)) inhibited platelet accumulation and fibrin deposition in wild-type mice. Infusion of ERp72(oo-ss-ss) into β3-null mice increased fibrin deposition in the absence of platelets. ERp72-null platelets had defective aggregation, JON/A binding, P-selectin expression, and adenosine triphosphate (ATP) secretion. The aggregation and ATP secretion defects were fully rescued by ERp72(oo-ss-ss) but partially rescued by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). Aggregation and ATP secretion of human platelets was potentiated by ERp72(oo-ss-ss) but inhibited by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). These data suggest that both the a and a' active sites are required for platelet function. ERp72 bound poorly to β3-null mouse platelets, and the addition of ERp72(oo-ss-ss) to human platelets generated thiols in αIIbβ3, suggesting a direct interaction of ERp72 with αIIbβ3. Defective aggregation of ERp72-null platelets was recovered by ERp72, but not other thiol isomerases. In summary, ERp72 plays a critical role in platelet function and coagulation through the a and a' CGHC motifs., (© 2017 by The American Society of Hematology.)

Platelets play a major role in the complex interactions involved in blood coagulation via multiple mechanisms. As reported in this issue, Schoergenhofer et al. tested the hypothesis that platelet inhibition by prasugrel, a potent platelet P2Y12 ADP receptor antagonist, attenuates the effect of lipopolysaccharide (LPS) on the blood coagulation system in healthy human subjects. LPS, a bacterial product with potent pro-inflammatory and pro-thrombotic effects, plays a central role in sepsis. It activates monocytes and endothelial cells via Toll-like receptor (TLR) 4 and other TLRs to stimulate production of TF and other pro-coagulant molecules, chemokines and cytokines. Treatment with prasugrel did not decrease biomarkers of coagulaion. A better understanding of the relative roles of platelet and coagulation mechanisms in triggering the pro-thrombotic state may lead to more effective antithrombotic strategies., (© 2016 Authors; published by Portland Press Limited.)

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation.

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., (© 2014 International Society on Thrombosis and Haemostasis.)

The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbβ3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking β3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbβ3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

A close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated 2 rabbit Abs to ERp57. One Ab strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross-reactivity of this Ab with PDI by Western blot or in the functional assay. This Ab substantially inhibited activation of the αIIbβ3 fibrinogen receptor and P-selectin expression. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When infused into mice the inactive ERp57 prolonged the tail bleeding times. We generated 2 IgG2a mAbs that reacted with ERp57 by immunoblot. One of these Abs inhibited both ERp57 activity and platelet aggregation. The other Ab did not inhibit ERp57 activity or platelet aggregation. The inhibitory Ab inhibited activation of αIIbβ3 and P-selectin expression, prolonged tail bleeding times, and inhibited FeCl(3)-induced thrombosis in mice. Finally, we found that a commonly used mAb to PDI also inhibited ERp57 activity. We conclude that a glycoprotein-specific member of the PDI family, ERp57, is required for platelet aggregation, hemostasis, and thrombosis.

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 β3 of the αIIbβ3 fibrinogen receptor were required for platelet aggregation., Methods and Results:  Using p-chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbβ3 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 β3. 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 β-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 αIIbβ3. 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 αIIbβ3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non-labeled αIIbβ3 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 αIIbβ3 provides redox sensitive sites for regulation of αIIbβ3., (© 2011 International Society on Thrombosis and Haemostasis.)

There has recently been a dramatic expansion in research in the area of redox biology with systems that utilize thiols to perform redox chemistry being central to redox control. Thiol-based reactions occur in proteins involved in platelet function, including extracellular platelet proteins. The alphaIIbbeta3 fibrinogen receptor contains free thiols that are required for the activation of this receptor to a fibrinogen-binding conformation. This process is under enzymatic control, with protein disulfide isomerase playing a central role in the activation of alphaIIbbeta3. Other integrins, such as the alpha2beta1 collagen receptor on platelets, are also regulated by protein disulfide isomerase and thiol metabolism. Low molecular weight thiols that are found in blood regulate these processes by converting redox sensitive disulfide bonds to thiols and by providing the appropriate redox potential for these reactions. Additional mechanisms of redox control of platelets involve nitric oxide that inhibits platelet responses, and reactive oxygen species that potentiate platelet thrombus formation. Specific nitrosative or oxidative modifications of thiol groups in platelets may modulate platelet function. Since many biologic processes are regulated by redox reactions that involve surface thiols, the extracellular redox state can have an important influence on health and disease status and may be a target for therapeutic intervention.

The platelet integrin alpha(IIb)beta(3) mediates the final step of platelet aggregation that requires pre-activation through an inside-out signal initiated by agonists. Experiments conducted under static conditions using platelet-rich plasma show that platelet activation and adhesion activity of alpha(IIb)beta(3) are regulated by glutathione (GSH-GSSG) redox potential. However, it remains unclear as to whether GSH-GSSG exerts its regulatory role in platelets by direct targeting of alpha(IIb)beta(3) or intracellular signals that activate the integrin. A role of fluid shear stress is also not known. We examined the effects of GSH-GSSG on the adhesion of CHO cells expressing two HPA variants of human alpha(IIb)beta(3) to the immobilized fibrinogen and von Willebrand factor (VWF) under flow conditions. GSH-GSSG dose-dependently reduced the number of adherent cells to fibrinogen and VWF under 2.5 dyn/cm(2) of shear stress, a physical force calculated to be 110 dyne on platelets. GSH treatment also abolished the hyper-adhesion activity of cells expressing the Pro33 variant of alpha(IIb)beta(3). The inhibition was also observed with washed platelets. The data differ from the early observation that GSH enhanced platelet aggregation induced by sub-threshold concentrations of platelet agonists. The results suggest that GSH may have distinct effects on agonist-induced alpha(IIb)beta(3) activation and on the alpha(IIb)beta(3)-fibrinogen or alpha(IIb)beta(3)-VWF bonds when exposed to fluid shear stress. They further suggest that the HPA phenotype may be redox-regulated.

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

Platelet protein disulphide isomerase (PDI) has a role in platelet aggregation, probably targeting a thiol-containing platelet surface protein. The thiol-containing P2Y(12) ADP receptor is involved in aggregation induced by most agonists and may be the target of PDI. By excluding the P2Y(12) pathway and using the anti-PDI antibody RL90 this study showed that PDI targets a non-P2Y(12) thiol-protein in aggregation. Anti-PDI inhibited signalling-independent activation of the thiol-containing fibrinogen receptor alphaIIbbeta3 by Mn(2+), suggesting that PDI directly interacts with alphaIIbbeta3. The thiol-containing form of PDI increased on the platelet surface with platelet activation, suggesting that active PDI readily becomes available for redox regulation of alphaIIbbeta3. Finally, using purified proteins PDI had greater ability to isomerize disulphide bonds than the alphaIIbbeta3 integrin, which also has PDI-like activity. In summary, a mechanism exists in platelets to increase the functional form of surface PDI and this PDI has a non-P2Y(12) target that may be alphaIIbbeta3.

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.

We previously found that reduced glutathione (GSH) or a mixture of GSH/glutathione disulfide (GSSG) potentiated platelet aggregation. We here report that GSSG, when added to platelets alone, also potentiates platelet aggregation. Most of the GSSG was converted to GSH by a flavoprotein-dependent platelet surface mechanism. This provided an appropriate redox potential for platelet activation. The addition of GSSG to platelets generated sulfhydryls in the beta subunit of the alpha(IIb)beta(3) fibrinogen receptor, suggesting a mechanism for facilitation of agonist-induced platelet activation.

Disulfide bonds formed in newly synthesized proteins in the endoplasmic reticulum of cells are important for protein structure and stability. Recent research, however, emphasizes a role for thiol-disulfide reactions with disulfide bond rearrangement as a dynamic process in cell and protein function, and in platelet function in particular. Protein disulfide isomerase was found on the platelet surface where it appears to play an important role in the platelet responses of aggregation and secretion, as well as activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. Additionally, sulfhydryl groups in alphaIIbbeta3 have been implicated in the activation of this integrin. Physiologic concentrations of reduced glutathione generate sulfhydryls in alphaIIbbeta3 and potentiate sulfhydryl-dependent reactions in alphaIIbbeta3. Sulfhydryl labeling in alphaIIbbeta3 is inhibited by phenylarsine oxide, a reagent that binds to vicinal thiols. As vicinal thiols are in equilibrium with disulfide bonds, they provide redox-sensitive sites in alphaIIbbeta3 able to respond to external or cytoplasmic reducing equivalents. Furthermore, protein disulfide isomerase and sulfhydryls are now implicated in platelet adhesion by a second platelet integrin, the alpha2beta1 collagen receptor. Most recently, extracellular sulfhydryls in the P2Y12 ADP receptor were found to be required for platelet activation by this receptor. We here provide an overview of this field with a focus on recent developments, and conclude with a working model.

Integrin alpha2beta1 is the principal adhesive receptor for collagen but platelets also adhere through glycoprotein VI (GPVI). Integrin alphaIIbbeta3 may augment platelet adhesion. We have shown that disulfide exchange is necessary for platelet adhesion to fibrinogen, fibronectin, and collagen. However 2 questions remained: (1) Can activated alphaIIbbeta3 explain the observed role of disulfide exchange in adhesion to collagen, or is this role common to other integrins? (2) Is disulfide dependence specific to the integrin receptors or shared with GPVI? To discriminate adhesive functions of alpha2beta1 from those of alphaIIbbeta3 we used Glanzmann platelets and alphaIIbbeta3-specific antibodies applied to normal platelets. To resolve adhesive events mediated by alpha2beta1 from those of GPVI we used synthetic peptides specific to each receptor. We addressed direct integrin ligation using purified alpha2beta1 and recombinant I domain. We observed the following: adhesion to the alpha2beta1-specific peptide was disulfide-exchange dependent and protein disulfide isomerase (PDI) mediated; membrane-impermeant thiol blockers inhibited alpha2beta1, but not GPVI mediated, adhesion; direct blockade of PDI revealed that it is involved in adhesion through alpha2beta1 but not GPVI; and purified alpha2beta1, but not recombinant I domain, depended on free thiols for ligation. These data suggest that the enzymatically catalyzed adhesion-associated reorganization of disulfide bonds is common to members of the integrin family and specific to this family.

Sulfhydryl and disulfide metabolism in platelet function has recently reemerged as a focus of platelet research. In this study we tested the effect of redox buffer on platelet aggregation and the effect of reduced glutathione (GSH) and platelet activation on sulfhydryl exposure in the platelet fibrinogen receptor, alpha IIb beta 3. In the presence of subthreshold concentrations of agonist, physiologic concentrations of GSH (10 microM) stimulated platelet aggregation and secretion. These effects were found with more than one platelet agonist and with different low molecular weight thiols, including homocysteine. The effect of low molecular weight thiols was reproduced with the peptide LSARLAF which directly activates platelets through alpha IIb beta 3, suggesting that the mechanism is at the level of this integrin. After determining optimal sulfhydryl labeling conditions for alpha IIb beta 3 (5 mM EDTA, 37 degrees C, 60 min), we found that GSH (10 microM) generated sulfhydryls in the beta 3 subunit. To determine if the requirement was for reducing equivalents or for a redox potential (ratio of GSH to GSSG), aggregation was further studied with the addition of low concentrations of GSSG to the GSH. With a ratio of GSH/GSSG of 5/1, similar to that of blood, the addition of GSSG potentiated the stimulatory effect as compared to GSH alone. This indicates that, for potentiation of aggregation, GSH is not simply reducing disulfide bonds; there is rather a requirement for a certain redox potential. Additional studies performed in the absence of added glutathione showed an increase in sulfhydryl labeling in the beta 3 subunit during platelet activation. Finally, we show that vicinal dithiols of platelet surface proteins are involved in the sulfhydryl-dependent pathways of platelet activation. In summary, these data imply that the redox potential of blood regulates activation of the alpha IIb beta 3 integrin and together with other reports in the literature suggest that disulfide bond cleavage with sulfhydryl generation in beta 3 is involved in activation of this receptor.

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 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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

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 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.

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.

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.

Community Mental Health Centers often include the client in evaluation of services through the use of comprehensive and lengthy client satisfaction surveys. However, the results are often uniformly positive across the many issues on the survey, suggesting that many items are redundant. A comprehensive client satisfaction survey form containing 33 items was developed and administered to 170 terminated clients of three mental health agencies. A factor analysis of the data revealed dimensions of Satisfaction with Services, Acceptability of Clinician, Impact of Services, and Dignified Treatment. Demographic variables were not correlated with the factors. The original survey was reduced to 10 items and a Comments block. The 10 items are representative of the four factors and also included selected aspects of the agencies' programs and administration. The revised form can be used in a wide range of mental health settings.