The Jeremiah Metzger Lecture. Hypercoagulable states: challenges and opportunities.

Thromboregulatory physiology is essentially a function of the blood vessel wall. Constitutive endothelial cell activities maintain blood fluidity by down regulating the initiation as well as, the propagation of blood coagulation. The major systems involved include: the Protein C, TFPI, plasmin generating and antithrombin pathways, all of which are focused on the cell membrane. Altered regulation of these endothelial functions forms the basis of the pathophysiologic events associated with the inherited primary hypercoagulable states. Secondary hypercoagulable syndromes occurring in various clinical states with conversion to a vascular thrombogenic phenotype reflect non constitutive activated endothelial cell functions with concomitant down regulation of the constitutive anticoagulant surface activity. So called idiopathic clinical thrombosis in most circumstances represents multi hit events in which specific genetic abnormalities or polymorphisms together with specific acquired alterations in geographically distinct endothelial cell beds culminate in a recognizable coagulation phenotype.

Histidine-rich glycoprotein inhibits the antiangiogenic effect of thrombospondin-1.

Angiogenesis is critical for the growth and proliferation of tumors as well as for normal development. We now describe a novel role for histidine-rich glycoprotein (HRGP) in the modulation of angiogenesis. HRGP is a plasma protein that circulates in relatively high concentrations (1.5 microM), but has no known function in vivo. We have shown previously that HRGP binds with high affinity to thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that is a potent inhibitor of angiogenesis. The antiangiogenic activity of TSP-1 is mediated by the binding of properdin-like type I repeats to the receptor CD36. We found that binding of HRGP to TSP-1 was similarly mediated by TSP type I repeats. HRGP colocalized with TSP-1 in the stroma of human breast cancer specimens, and this interaction masked the antiangiogenic epitope of TSP-1. In assays performed in vitro of endothelial cell migration and tube formation, and in vivo corneal angiogenesis assays, HRGP inhibited the antiangiogenic effect of TSP-1. These studies suggest that HRGP can modulate the antiangiogenic activity of TSP-1, and identify a potential mechanism of resistance to the antiangiogenic effect of TSP-1.

Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation.

Although thrombopoietin has been shown to promote megakaryocyte (MK) proliferation and maturation, the exact mechanism and site of platelet formation are not well defined. Studies have shown that MKs may transmigrate through bone marrow endothelial cells (BMEC), and release platelets within the sinusoidal space or lung capillaries. In search for chemotactic factor(s) that may mediate transmigration of MKs, we have discovered that mature polyploid MKs express the G protein-coupled chemokine receptor CXCR4 (Fusin, LESTR). Therefore, we explored the possibility that stromal cell-derived factor 1 (SDF-1), the ligand for CXCR4, may also induce transendothelial migration of mature MKs. SDF-1, but not other CXC or CC chemokines, was able to mediate MK migration (ED50 = 125 pmol/liter). The MK chemotaxis induced by SDF-1 was inhibited by the CXCR4-specific mAb (12G5) and by pertussis toxin, demonstrating that signaling via the G protein-coupled receptor CXCR4 was necessary for migration. SDF-1 also induced MKs to migrate through confluent monolayers of BMEC by increasing the affinity of MKs for BMEC. Activation of BMEC with interleukin 1beta resulted in a threefold increase in the migration of MKs in response to SDF-1. Neutralizing mAb to the endothelial-specific adhesion molecule E-selectin blocked the migration of MKs by 50%, suggesting that cellular interaction of MKs with BMEC is critical for the migration of MKs. Light microscopy and ploidy determination of transmigrated MKs demonstrated predominance of polyploid MKs. Virtually all platelets generated in the lower chamber also expressed CXCR4. Platelets formed in the lower chamber were functional and expressed P-selectin (CD62P) in response to thrombin stimulation. Electron microscopy of the cells that transmigrated through the BMEC monolayers in response to SDF-1 demonstrated the presence of intact polyploid MKs as well as MKs in the process of platelet formation. These results suggest that SDF-1 is a potent chemotactic factor for mature MKs. Expression of CXCR4 may be the critical cellular signal for transmigration of MKs and platelet formation.

Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.

Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.

Transendothelial migration of CD34+ and mature hematopoietic cells: an in vitro study using a human bone marrow endothelial cell line.

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 microns microporous membrane inserts and placed in 6-well tissue culture plates. Granulocytecolony stimulating factor (G-CSF)-mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 +/- 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 +/- 0.1 v 7.8% +/- 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% +/- 0.8% v 0.3% +/- 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% +/- 0.4%). While no difference in the expression of the beta 1-integrin very late activation antigen (VLA)-4 and beta 2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics.

Bone marrow microvascular endothelial cells (BMEC) are a functional component of the bone marrow stroma and have been shown to release hematopoietic regulatory factors as well as to selectively adhere and support the proliferation and differentiation of CD34+ hematopoietic progenitors. An early passage of these cells was immortalized by transfection with a vector (pSVT) encoding the large T antigen of SV40. The transformed cell line (CDC/CU.BMEC-1) expresses the SV40 transcript, retains the primary cell expression of Ulex europeaus and vWF/ FVIII, and incorporates acetylated low-density lipoprotein. In addition, BMEC-1 mirrors the phenotype of the primary cells with only a few exceptions. Both cell populations express the cellular adhesion molecules ICAM-1 and PECAM and also VCAM-1 and ELAM-1 after upregulation by tumor necrosis factor-alpha. The fibronectin receptor, hyaluronate receptor, collagen receptor, integrins VLA-alpha 3, VLA-alpha 4, and beta 4, endoglin, collagen IV, CD58, and CD61 are also expressed. The only differences are that BMEC-1 expresses higher levels of ICAM-1, CD58, CD34, CD36, and c-kit than the primary cells. The supernatants of primary cell and BMEC-1 contain stem cell factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-11, and G-CSF. The functional significance of these hematopoietic cytokines was demonstrated in transwell cultures. Both cell populations supported the expansion of progeny from CD34+ cell-enriched cord blood mononuclear cells suspended in the upper chamber. These characteristics, plus the fact that BMEC-1 can be maintained independently of exogenous growth factors and exhibit contact inhibition, indicate that this cell line can be used to further define the role of BMEC in hematopoiesis.

Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa.

The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196 --> Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.

The role of lipoprotein(a) in atherogenesis and thrombosis.

Lipoprotein(a) [Lp(a)] represents an important independent risk factor for atherosclerotic cardiovascular disease. Lp(a) constitutes a class of low-density lipoprotein-like particles that are structurally heterogeneous due to variability within the distinguishing apoprotein, apolipoprotein(a) [Apo(a)]. Apo(a) bears a high degree of homology to the fibrinolytic zymogen, plasminogen, the parent molecule of the serine protease plasmin. Apo(a) contains a variable number of tandemly repeated triple-loop units called kringles, which appear to mediate Lp(a)'s interactions with fibrin and cell surface receptors. Although the mechanism of its atherogenicity is unknown, Lp(a) has been implicated in the delivery of cholesterol to the injured blood vessel, in blockade of plasmin generation on fibrin and cell surfaces, and as a stimulus for smooth muscle cell proliferation. In addition, new members of the plasminogen/Apo(a) gene family have been defined, creating a potential link between Lp(a) and the control of angiogenesis in both health and disease. Pharmacologic therapy of elevated Lp(a) levels has been only modestly successful; apheresis remains the most effective therapeutic modality.

Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices.

BACKGROUND: Textured biomaterial surfaces in implantable left ventricular assist devices induce development of a nonthrombotic neointimal surface and allow elimination of anticoagulation therapy in device recipients. Characterization of the hematopoietic cells formed within the neointimal surfaces of these devices will contribute to our understanding of this unique neointima. METHODS: The blood-contacting surface of seven ThermoCardiosystems left ventricular assist devices was removed, washed with phosphate-buffered saline solution, and digested with 0.1% collagenase for 15 to 20 minutes. The hematopoietic cells released from the explants were isolated and analyzed by flow cytometry and immuno-histochemical staining. RESULTS: More than 80% +/- 6% of hematopoietic cells isolated in this fashion are of myelomonocytic origin and express CD14, CD15, and CD33 surface molecules. Four percent of cells express the CD34 surface marker, which suggests that the neointima is colonized by pluripotent hematopoietic stem cells. Continuous culture of these hematopoietic cells in the presence of the cytokines interleukin-3, c-kit ligand, granulocyte colony-stimulating factor resulted in tenfold expansion by day 7 and 25-fold expansion by day 14. CONCLUSIONS: Pluripotent hematopoietic cells with a high proliferative capacity colonize textured surfaces of left ventricular assist devices and may contribute to the development of a biologically nonthrombogenic neointima.

Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors.

Endothelial cells are a major component of the bone marrow (BM) microenvironment that regulate the trafficking and homing of hematopoietic progenitor and stem cells. In this paper, we provide evidence that BM endothelial cells (BMECs) also support multilineage hematopoiesis by elaboration of soluble cytokines. Hematopoietic progenitor cells incubated in direct contact with BMEC monolayers, or physically separated by microporous membrane, expanded five-fold to sevenfold at 7 days, in the absence of exogenous cytokines. Flow cytometric analysis of proliferating progenitor cells grown in the presence of BMEC monolayers showed that by day 14 of coculture, 70% to 80% of hematopoietic cells were myeloid, expressing CD15 or CD14, and 14% to 19% were megakaryocytic, expressing GPIIb/IIIa or GPIb. CD34+ cells derived from umbilical cord blood, cultured in the upper chamber of transwell culture plates, as well as the cells grown in direct contact with BMEC monolayers, generated progenitors for up to 70 days. Unstimulated BMEC monolayers constitutively produce interleukin-6, Kit-ligand, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. These data suggest that BMEC regulate proliferation of hematopoietic progenitor cells and long-term culture initiating cells by elaboration of lineage-specific cytokines.

Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion.

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.

Hypercoagulable states.

PURPOSE: To describe the major pathophysiologic mechanisms underlying inherited and secondary hypercoagulable states and to evaluate the frequency, natural history, diagnosis, and management of the various clinical disorders. DATA SOURCES AND STUDY SELECTION: Relevant clinical literature obtained from bibliographies in hematology textbooks and from computerized indexes was reviewed. A hypothesis was formed based on this literature review and on recent developments from a number of experimental studies. DATA SYNTHESIS: Hypercoagulable states include various inherited as well as acquired clinical disorders characterized by an increased risk for thromboembolism. Primary hypercoagulable states include relatively rare inherited conditions that lead to disordered endothelial cell thromboregulation. These conditions include decreased thrombomodulin-dependent activation of activated protein C, impaired heparin binding of antithrombin III, or down-regulation of membrane-associated plasmin generation. The major, inherited, inhibitor disease states include antithrombin III deficiency, protein C deficiency, and protein S deficiency and should be considered in patients who have recurrent, familial, or juvenile deep-vein thrombosis or occlusion in an unusual location such as a mesenteric, brachial, or cerebral vessel. Secondary hypercoagulable states may be seen in many heterogeneous disorders. In many of these conditions, endothelial activation by cytokines leads to loss of normal vessel-wall anticoagulant surface functions with conversion to a proinflammatory thrombogenic phenotype. Important clinical syndromes associated with substantial thromboembolic events include the antiphospholipid syndrome, heparin-induced thrombopathy, the myeloproliferative syndromes, and cancer. CONCLUSIONS: Physiologic thromboregulation occurs at the vessel-wall surface. Quantitative and qualitative deficiencies of normal, steady-state endothelial anticoagulant activities are associated with primary hypercoagulable states. Activated endothelial cell surfaces express a thrombogenic phenotype and contribute to secondary or acquired hypercoagulability.

Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.

Lipoprotein (a) regulates plasminogen activator inhibitor-1 expression in endothelial cells. A potential mechanism in thrombogenesis.

Lipoprotein (a) (Lp(a)) is a low density lipoprotein-like particle which contains the plasminogen-like apolipoprotein a. Lp(a) levels are elevated in patients with atherosclerotic coronary artery disease. Recent studies suggest that Lp(a) competitively inhibits plasminogen binding to the endothelial cell and interferes with surface-associated plasmin generation. In this study, we present evidence for the presence of Lp(a) in the microvasculature of inflamed tissue. In addition, we demonstrate that Lp(a) regulates endothelial cell synthesis of a major fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). In cultured human endothelial cells, Lp(a) enhanced PAI-1 antigen, activity, and steady-state mRNA levels without altering tissue plasminogen activator activity or mRNA transcript levels. This effect was cell-specific. Although other lipoproteins did not coordinately raise PAI-1 mRNA levels in endothelial cells, low density lipoprotein treatment selectively raised the level of the 3.4-kilobase mRNA species of PAI-1 without a concomitant increase in PAI-1 activity or antigen. Endothelial cell exposure to Lp(a) did not cause generalized endothelial cell activation since the functional activity and mRNA levels for tissue factor, platelet-derived growth factor and interleukin-6 were not elevated following Lp(a) exposure. These data suggest a molecular mechanism whereby Lp(a) may support a specific prothrombotic endothelial cell phenotype, namely by increasing PAI-1 expression.