Combined immunodeficiency (CVID and CD4 lymphopenia) is associated with a high risk of malignancy among adults with primary immune deficiency

Primary immunodeficiency disorders (PID) are a group of heterogeneous disorders characterized by recurrent infections, autoimmunity, increased lymphoproliferative disorders and other malignancies. PID is classified into cellular or humoral disorders or a combination of both. We evaluated the clinical differences among adult patients with three variants of PID: common variable immunodeficiency (CVID), idiopathic CD4 lymphopenia (ICL) and combined immunodeficiency (CID). We retrospectively compared demographics, immunological characteristics, clinical presentations and outcomes of CVID, CID and ICL patients followed from 2012 to 2018. In our cohort, we identified 44 adult patients diagnosed with CVID (22), CID (11) and ICL (11). Malignancy was associated with CID, as seven of 11 patients in this group were diagnosed with malignancy compared to CVID (three of 22) or ICL (two of 11) (P = 0·002 and 0·03, respectively). Malignancies were also linked to male gender [odds ratio (OR) = 5, 95% confidence interval (CI) = 1·12-22·18) P = 0·0342] and a low ratio of CD4/CD8 < 0·8 (OR = 5·1, 95% CI = 1·22-21·28, P = 0·025). Among CID and ICL, two of 11 patients died in each group, while no death was documented among CVID group (P = 0·04). Autoimmune manifestations did not differ between groups. Similarly, the rate of infections was similar between groups, although infectious agents vary. CID is associated with a high risk of malignancy compare to CVID or ICL. Among adults with PID, male gender, low CD4 and a CD4/CD8 ratio of < 0·8 may serve as risk factors of concomitant malignancy. Surveillance of lymphocyte subpopulations should be considered for all adults.

Exploring the metabolic variation between domesticated and wild tetraploid wheat genotypes in response to corn leaf aphid infestation.

Infestation of Triticum (wheat) plants by their pest Rhopalosiphum maidis (corn leaf aphid) causes severe vegetative damage. Despite the agro-economic importance of wheat, the metabolic diversity of Triticum turgidum (tetraploid wheat) in response to aphid attack has not been sufficiently addressed. In this study, we compared the metabolic diversity of two tetraploid wheat genotypes, domesticated and wild emmer. The plants were grown in a control growth room and infested with aphids for 96 h. Our untargeted metabolic analysis performed on plants with and without aphids revealed massive differences between the two genotypes. The targeted metabolic analysis highlighted the differences in the biosynthesis of phytohormones. The aphid progeny was lower in the cultivated durum wheat than in the wild emmer wheat. Overall, these observations emphasize the potential of using the natural diversity of wheat species to better understand the metabolic responses to pest damage.

Protease-activated receptors (PARs) in cancer: Novel biased signaling and targets for therapy.

Despite the fact that G protein-coupled receptors (GPCRs) mediate numerous physiological processes and represent targets for therapeutics for a vast array of diseases, their role in tumor biology is under appreciated. Protease-activated receptors (PARs) form a family which belongs to GPCR class A. PAR1&2 emerge with a central role in epithelial malignancies. Although the part of PAR1&2 in cancer is on the rise, their underlying signaling events are poorly understood. We review hereby past, present, and future cancer-associated PAR biology. Mainly, their role in physiological (placenta-cytotophobalst) and patho-physiological invasion processes. The identification and characterization of signal pleckstrin homology (PH)-domain-binding motifs established critical sites for breast cancer growth in PAR1&2. Among the proteins found to harbor important PH-domains and are involved in PAR biology are Akt/PKB as also Etk/Bmx and Vav3. A point mutation in PAR2, H349A, but not R352A, abrogated PH-protein association and is sufficient to markedly reduce PAR2-instigated breast tumor growth in vivo as also placental extravillous trophoblast (EVT) invasion in vitro is markedly reduced. Similarly, the PAR1 mutant hPar1-7A, which is unable to bind PH-domain, inhibits mammary tumors and EVT invasion, endowing these motifs with physiological significance and underscoring the importance of these previously unknown PAR1 and PAR2 PH-domain-binding motifs in both pathological and physiological invasion processes.

PH motifs in PAR1&2 endow breast cancer growth.

Although emerging roles of protease-activated receptor1&2 (PAR1&2) in cancer are recognized, their underlying signalling events are poorly understood. Here we show signal-binding motifs in PAR1&2 that are critical for breast cancer growth. This occurs via the association of the pleckstrin homology (PH) domain with Akt/PKB as a key signalling event of PARs. Other PH-domain signal-proteins such as Etk/Bmx and Vav3 also associate with PAR1 and PAR2 through their PH domains. PAR1 and PAR2 bind with priority to Etk/Bmx. A point mutation in PAR2, H349A, but not in R352A, abrogates PH-protein association and is sufficient to markedly reduce PAR2-instigated breast tumour growth in vivo and placental extravillous trophoblast (EVT) invasion in vitro. Similarly, the PAR1 mutant hPar1-7A, which is unable to bind the PH domain, reduces mammary tumours and EVT invasion, endowing these motifs with physiological significance and underscoring the importance of these previously unknown PAR1 and PAR2 PH-domain-binding motifs in both pathological and physiological invasion processes.

Topological properties of microwave magnetoelectric fields.

Collective excitations of electron spins in a ferromagnetic sample dominated by the magnetic dipole-dipole interaction strongly influence the field structure of microwave radiation. A small quasi-two-dimensional ferrite disk with magnetic-dipolar-mode (MDM) oscillation spectra can behave as a source of specific fields in vacuum, termed magnetoelectric (ME) fields. A coupling between the time-varying electric and magnetic fields in the ME-field structures is different from such a coupling in regular electromagnetic fields. The ME fields are characterized by strong energy confinement at a subwavelength region of microwave radiation, topologically distinctive power-flow vortices, and helicity parameters [E. O. Kamenetskii, R. Joffe, and R. Shavit, Phys. Rev. E 87, 023201 (2013)]. We study topological properties of microwave ME fields by loading a MDM ferrite particle with different dielectric samples. We establish a close connection between the permittivity parameters of dielectric environment and the topology of ME fields. We show that the topology of ME fields is strongly correlated with the Fano-resonance spectra observed at terminals of a microwave structure. We reveal specific thresholds in the Fano-resonance spectra appearing at certain permittivity parameters of dielectric samples. We show that ME fields originated from MDM ferrite disks can be distinguished by topological portraits of the helicity parameters and can have a torsion degree of freedom. Importantly, the ME-field phenomena can be viewed as implementations of space-time coordinate transformations on waves.

Microwave magnetoelectric fields and their role in the matter-field interaction.

We show that in a source-free subwavelength region of microwave fields, there can exist field structures with a local coupling between the time-varying electric and magnetic fields differing from the electric-magnetic coupling in regular-propagating free-space electromagnetic waves. To distinguish such field structures from regular electromagnetic (EM) field structures, we term them as magnetoelectric (ME) fields. We study a structure and conservation laws of microwave ME near fields. We show that there exist sources of microwave ME near fields-the ME particles. These particles are represented by small quasi-two-dimensional ferrite disks with magnetic-dipolar-oscillation spectra. The near fields originating from such particles are characterized by topologically distinctive power-flow vortices, nonzero helicity, and a torsion degree of freedom. The paper consists of two main parts. In the first one, we give a theoretical background of properties of the electric and magnetic fields inside and outside of a ferrite particle with magnetic-dipolar-oscillation spectra resulting in the appearance of microwave ME near fields. In the second main part, we represent numerical and experimental studies of the microwave ME near fields and their interactions with matter. Based on the obtained properties of the ME near fields, we discuss possibilities for effective microwave sensing of natural and artificial chiral structures.

Magnetic-dipolar and electromagnetic vortices in quasi-2D ferrite discs.

Magnetic-dipolar-mode (MDM) oscillations in a quasi-2D ferrite disc show unique dynamical symmetry properties resulting in the appearance of topologically distinct structures. Based on the magnetostatic (MS) spectral problem solutions, in this paper we give evidence for eigen-MS power-flow-density vortices in a ferrite disc. Due to these circular eigen-power flows, the MDMs are characterized by MS energy eigenstates. It becomes evident that the reason for stability of the vortex configurations in saturated ferrite samples is completely different from the nature of stability in magnetically soft cylindrical dots. We found a clear correspondence between analytically derived MDM vortex states and numerically modeled electromagnetic vortices in quasi-2D ferrite discs.

p53 controls hPar1 function and expression.

Human protease-activated receptor 1 (hPar1) is a bona fide receptor of the hemostatic protease thrombin, and has a central function in tumor progression. Inactivation of the tumor suppressor gene p53 is one of the most common genomic alterations occurring in cancer. Here, we address the interrelations between p53 and hPar1 in cancer. We demonstrate an inverse correlation between hPar1 gene expression and wild-type (wt) p53 levels, and a direct correlation with levels of the mutant (mt) p53. Bioinformatic search revealed the presence of at least two p53 motifs in the hPar1 promoter. Indeed, temperature-sensitive (ts) p53 forms reduced hPar1 promoter activity on wt p53 expression. Ectopic introduction of the p53R175H mutant into cells lacking p53 caused a moderate two-fold induction of hPar1 promoter activity. Chromatin immunoprecipitation (ChIP) analyses confirmed a physical association between the p53 protein and hPar1 chromatin fragments. In parallel, PAR1 function is attenuated by p53, as shown by inhibition of pFAK levels and a Matrigel invasion assay. Ectopic reinforcement of hPar1 rescued the inhibition conferred by p53, confirming that p53 directly affects hPar1 expression and function. Altogether, we provide evidence for a direct binding between p53 and hPar1 chromatin, and assign hPar1 as a target of p53.

Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin.

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.

Protection of thrombin receptor expression under hypoxia.

Thrombin receptor (ThR) plays a significant role in myocyte contractility and hypertrophy. Heart myocyte ischemic damage, caused by insufficient blood supply, is the leading cause of heart infarction. Here we demonstrate that when primary myocyte cultures are subjected to hypoxic stress, ThR mRNA levels are reduced markedly. This takes place also in vivo in a model of ischemic pig heart, exhibiting reduced levels of ThR compared with normal heart sections. Prior activation of ThR however, by either thrombin receptor-activating peptide (TRAP) or by alpha-thrombin resulted in full protection of ThR mRNA levels under hypoxia. The effect appeared specific to ThR because the addition of TRAP did not affect the hypoxic damage as shown by the levels of lactic dehydrogenase release and up-regulated GLUT-1, a glucose transporter gene. This protection effect took place not only in primary myocytes but also in NIH3T3 fibroblasts. ThR protection occurs via specific cell signaling events because activation of the receptor by TRAP, following interruption of the signaling cascade by calphostin C, a protein kinase C inhibitor, resulted in loss of ThR mRNA protection. Because Ras and Src are part of the ThR signaling cascade, the introduction of either dominant ras or src oncogenes to NIH3T3 murine fibroblasts gave rise to similar protection of ThR mRNA levels under hypoxic conditions without the exogenous addition of TRAP. Likewise, ThR mRNA protection was obtained after transfection with proto-oncogene vav. The 95-kDa protein Vav undergoes tyrosine phosphorylation after ThR activation, serving thus as part of the receptor machinery cascade. We therefore conclude that the initiation of the signaling cascades either exogenously by TRAP or within the cell via src or ras, as well as via vav oncogene interconnecting G-binding protein to the tyrosine kinase pathway, ultimately results in ThR protection under hypoxia. We present hereby, a novel concept of activated receptors, which under minimal oxygen tension protect their otherwise decaying mRNA. Maintaining the level of ThR that plays an active role in normal myocyte function may provide a significant repair mechanism in ischemic tissue, assisting in the regaining of normal myocyte functions.

Thrombin receptor overexpression in malignant and physiological invasion processes.

Although the involvement of soluble and matrix-immobilized proteases in tumor cell invasion and metastasis is well recognized, the role of proteolytically activated cell surface receptors has not been elucidated. We report here that thrombin receptor, a member of the protease-activated receptor family, is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA considerably inhibited the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane. During placental implantation of the human embryo, thrombin receptor is transiently expressed in the invading cytotrophoblasts. These results emphasize the involvement of thrombin receptor in cell invasion associated with tumor progression and normal embryonic development.

Specific involvement of glypican in thrombin adhesive properties.

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.

Structural requirements for inhibition of melanoma lung colonization by heparanase inhibiting species of heparin.

Heparanase activity correlates with metastatic potentials of lymphoma, melanoma and mammary adenocarcinoma cell lines. We investigated the ability of various modified species of heparin and size homogeneous oligosaccharides derived from depolymerized heparin to inhibit: a) heparanase-mediated degradation of heparan sulfate (HS) in the extracellular matrix (ECM) deposited by cultured endothelial cells, and b) lung colonization of B16-BL6 melanoma cells in C57BL mice. For this purpose, melanoma cells or conditioned medium were incubated with metabolically sulfate-labeled subendothelial ECM in the absence and presence of heparin, heparin fragment or nonanticoagulant species of heparin. Labeled HS degradation fragments released into the incubation medium were analyzed by gel filtration over Sepharose 6B. The B16-BL6 melanoma cells were also tested for lung colonization following their intravenous administration to C57BL mice, in the absence and presence of the various species of heparin. Inhibition of both heparanase and melanoma lung colonization depended on the size and degree of sulfation of the heparin molecule, the position of sulfate groups, and the occupancy of the N position of the hexosamines. Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and O positions. Low sulfate oligosaccharides were less effective heparanase inhibitors than medium and high sulfate fractions of the same size saccharide. While O-desulfation abolished the heparanase inhibiting effect of heparin. O-sulfated, N-substituted (e.g., N-acetyl or N-hexanoyl) species of heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about > or = 4,000 daltons. Potent inhibitors of heparanase activity were also efficient inhibitors of tumor invasion and lung colonization. The antimetastatic and anticoagulant activities of heparin were unrelated, as indicated by using heparin fractions with high and low affinity for antithrombin III. These heparins differ about 200-fold in their anticoagulant activity, but expressed similar high antiheparanase and antimetastatic activities. It appears that heparanase-inhibiting species of heparin interfere with the passing of tumor cells across the capillary wall, as they significantly inhibited metastasis even when injected up to 3 h after lodgment. Structural requirements for inhibition of heparanase activity and lung colonization of melanoma cells by species of heparin were different from those identified for a) release of ECM-bound basic fibroblast growth factor (bFGF), and b) stimulation of bFGF receptor binding and mitogenic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The involvement of thrombin RGD in metastasis: characterization of a cryptic adhesive site.

Adhesive interactions between cells and the subendothelial extracellular matrix take place at several stages during tumor progression and metastasis. We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which can be exposed in the presence of low concentrations of plasmin and cell-associated heparan sulfate proteoglycan. Thus, thrombin may act as a matrix-adhesive molecule via activation of the alpha v beta 3 integrin. We have now identified a 31 amino acid fragment as the minimal thrombin-generated cleavage product, which contains an active RGD site, following gel filtration analysis on FPLC Superdex 75 column. The role of membrane-associated heparan sulfate in thrombin conversion to an adhesive protein was demonstrated by using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Incubation of both thrombin and a low concentration of plasmin on the surface of wild type CHO cells resulted in a typical digestion cleavage profile upon gel filtration. No cleavage products were observed when thrombin and a suboptimal plasmin concentration were incubated on monolayers of CHO cell mutants lacking heparan sulfate. Next, we examined the possible role of the thrombin RGD site during the progression of tumor development and metastasis. Toward this, we tested murine melanoma cells expressing low (B16-F1 cells) and high (B16-BL6 cells) lung colonization potentials in cell adhesion assays in vitro. Differential adherence capability of the cells was observed: while high attachment levels of B16-BL6 cells were obtained, the low metastatic B16-F1 cells did not adhere to thrombin RGD. Antibodies raised against the RGD site in thrombin specifically recognized thrombin digested with plasmin, but were unable to interact with native thrombin or prothrombin and inhibited potently B16-BL6 melanoma cell adhesion. Furthermore, the antibodies failed to recognize RGD in other adhesive plasma proteins such as vitronectin, fibrinogen, or fibronectin. Provided that the RGD-containing fragments of thrombin are widely distributed throughout the vascular system, they may have a significant role during tumor progression and dislodgement of metastatic cells. The development of RGD mimetics and/or specific antibodies might thus be applied to inhibit a critical step in metastatic spread.

Antiproliferative activity to vascular smooth muscle cells and receptor binding of heparin-mimicking polyaromatic anionic compounds.

Proliferation of bovine aortic smooth muscle cells (SMCs) induced by thrombin, basic fibroblast growth factor, or serum is inhibited by anionic, nonsulfated aromatic compounds that mimic many of the effects of heparin. Among these compounds are aurintricarboxylic acid (ATA) and a newly synthesized polymer of 4-hydroxyphenoxy acetic acid (compound RG-13577). Iodinated- or 14C-labeled compound RG-13577 binds to cultured SMCs in a highly specific and saturable manner. Scatchard analysis of the binding data revealed the presence of an estimated 1 x 10(7) binding sites per cell with an apparent dissociation constant of 3 x 10(-6) mol/L. Binding of radiolabeled RG-13577 was efficiently competed for by related aromatic anionic compounds and by apolipoprotein E, but not by heparin, heparan sulfate, suramin, or various purified growth factors and extracellular matrix proteins. Receptor cross-linking of SMC-bound 125I-RG-13577 revealed a single species of high M(r) (approximately 280 kD) cell surface receptors detected in the absence but not the presence of excess unlabeled compound RG-13577. Binding was susceptible to downregulation and restoration of receptor levels in a manner similar to that of hormone and growth factor receptors. We suggest that the antiproliferative activity of compound RG-13577 and related compounds is initiated by binding to specific growth-inhibiting cell surface receptors. Heparin-mimicking compounds may be applied to inhibit SMC proliferation associated with atherosclerosis and restenosis.

Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway.

Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin receptor, which both strongly reduced thrombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14-induced HUVEC proliferation was strongly inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HUVECs.

Thrombin adhesive properties: induction by plasmin and heparan sulfate.

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.

Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix.

The angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM-bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti-bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.