GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

Regulation of F-Actin Binding to Platelet Moesin In Vitro by Both Phosphorylation of Threonine 558 and Polyphosphatidylinositides

Activation of human platelets with thrombin transiently increases phosphorylation at 558threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.

A platelet–endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1

One crucial role of endothelium is to keep the innermost surface of a blood vessel antithrombotic. However, the endothelium also expresses prothrombotic molecules in response to various stimuli. The balance between the antithrombotic and prothrombotic nature of the endothelium is lost under certain conditions. During atherosclerosis, the attachment of platelets to the vessel surface has been suggested to promote the proliferation of smooth muscle cells and intimal thickening as well as to affect the prognosis of the disease directly through myocardial infarction and stroke. Dysfunctional endothelium, which is often a result of the action of oxidized low-density lipoprotein (OxLDL), tends to be more procoagulant and adhesive to platelets. Herein, we sought the possibility that the endothelial lectin-like OxLDL receptor-1 (LOX-1) is involved in the platelet–endothelium interaction and hence directly in endothelial dysfunction. LOX-1 indeed worked as an adhesion molecule for platelets. The binding of platelets was inhibited by a phosphatidylserine-binding protein, annexin V, and enhanced by agonists for platelets. These results suggest that negative phospholipids exposed on activation on the surface of platelets are the epitopes for LOX-1. Notably, the binding of platelets to LOX-1 enhanced the release of endothelin-1 from endothelial cells, supporting the induction of endothelial dysfunction, which would, in turn, promote the atherogenic process. LOX-1 may initiate and promote atherosclerosis, binding not only OxLDL but also platelets.

Blood-borne tissue factor: Another view of thrombosis

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused ≈70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VIIai, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor Xa formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.

Ex vivo evaluation of a Taylor-Couette flow, immobilized heparinase I device for clinical application

Efficient and safe heparin anticoagulation has remained a problem for continuous renal replacement therapies and intermittent hemodialysis for patients with acute renal failure. To make heparin therapy safer for the patient with acute renal failure at high risk of bleeding, we have proposed regional heparinization of the circuit via an immobilized heparinase I filter. This study tested a device based on Taylor-Couette flow and simultaneous separation/reaction for efficacy and safety of heparin removal in a sheep model. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. The device, referred to as a vortex flow plasmapheretic reactor, consisted of two concentric cylinders, a priming volume of 45 ml, a microporous membrane for plasma separation, and an outer compartment where the immobilized heparinase I was fluidized separately from the blood cells. Manual white cell and platelet counts, hematocrit, total protein, and fibrinogen assays were performed. Heparin levels were indirectly measured via whole-blood recalcification times (WBRTs). The vortex flow plasmapheretic reactor maintained significantly higher heparin levels in the extracorporeal circuit than in the sheep (device inlet WBRTs were 1.5 times the device outlet WBRTs) with no hemolysis. The reactor treatment did not effect any physiologically significant changes in complete blood cell counts, platelets, and protein levels for up to 2 hr of operation. Furthermore, gross necropsy and histopathology did not show any significant abnormalities in the kidney, liver, heart, brain, and spleen.

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzα also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giα family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.

A thrombin receptor function for platelet glycoprotein Ib–IX unmasked by cleavage of glycoprotein V

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V −/− platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib–IX–V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib–IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (Gq-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib–IX–V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib–IX via thrombin acting as a ligand, resulting in platelet activation.

Integrin activation controls metastasis in human breast cancer

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.

How the protease thrombin talks to cells

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1’s proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naïve receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.