Impact of different low-dose ritonavir regimens on lipids, CD36, and adipophilin expression.

We investigated the relationship between ritonavir concentrations and changes in lipids, vascular inflammation markers, CD36, and adipophilin expression in volunteers randomly assigned to groups receiving 100 mg of ritonavir once daily or twice daily. In both groups decreases in high-density lipoprotein (HDL) (6%, P = 0.010; and 10%, P < 0.001, respectively) and CD36 (14%, P = 0.012; and 16%, P = 0.006, respectively) and increases in the vascular inflammation marker sCD40L (12%, P = 0.008; 19%, P = 0.003, respectively) were seen. Increases in adipophilin (30%, P = 0.044) and triglycerides (32%, P = 0.044) were seen only in the group receiving ritonavir twice daily. The ritonavir concentration in the plasma correlated with changes in triglycerides, HDL, and adipophilin (r = 0.34, P = 0.030; r = 0.33, P = 0.040; and r = 0.4, P = 0.01, respectively).

Metabolic-inflammatory changes, and accelerated atherosclerosis in HIV patients: rationale for preventative measures.

Human immunodeficiency virus (HIV)-infected patients are at a significantly higher risk from coronary heart diseases (CHD) and myocardial infarction (MI) compared to gender- and age-matched non-infected individuals. Combination antiretroviral therapy (cART) has transformed a fatal illness into a chronic stable condition. However, cART induces metabolic abnormalities in HIV-infected patients, while its role in vascular atherosclerosis is still under investigation. The use of cART is linked to inflammation - a key mechanism in atherosclerotic progression and destabilisation that precedes clinical events like MI. There is evidence of visceral fat abnormal distribution in HIV infected patients, and inflammatory changes in HIV infected patients drive the initiation, progression and, ultimately, thrombotic clinical complications induced by atherosclerosis. Visceral adipose tissue, a virtual factory for manufacturing pro-inflammatory mediators, affects the liver function. The inflamed liver promotes the development of pro-atherogenic dyslipidaemia. Pro-inflammatory cytokines released by adipocytes travel to the skeletal muscles and other peripheral tissues, worsening insulin sensitivity and leading to hyperglycaemia. Increased high sensitivity C-reactive protein (hs-CRP) inflammatory marker is associated with endothelial dysfunction in HIV-infected patients. Increased levels of monocytic nuclear factor kappa-B (NFkappa-B), a master switch in the inflammatory cascade, are documented in patients with elevated hs-CRP levels. It can be assumed that, as a result of NFkappa-B activation, hs-CRP up-regulates cytokines that contribute to MI by recruiting leukocytes and promoting thrombosis. This review focuses on the association of HIV-infection, metabolic abnormalities and known mechanisms involved in inducing accelerated atherosclerosis and inflammation in HIV-infected patients, as well as the role of lipid lowering agents in potentially preventing CHD.

The dialogue between endothelial cells and monocytes/macrophages in vascular syndromes.

The aim of this chapter is to present and identify potential pharmacological targets in endothelial cell-monocyte interactions leading to vascular syndrome and involving inflammation, coagulation, vascular remodelling and thrombosis. Increasing evidence is indicating that endothelial cells play a key role in atherothombosis by their capacity to attract, bind and allow the extravasation of monocytes to sites of inflammation. Surface expression and/or activation of constituent cell adhesion molecules (for e.g. P-selectin, E-selectin, ICAM-1, and VCAM-1) on endothelial cells together with chemokines such as CXCL8 (IL-8), Platelet-activating factor (PAF), CCL2 and CCL5 (Table 1) allow the rolling, adhesion and extravasation of monocytes. This review focuses on pharmacological targets implicated in endothelial cells interactions with monocytes/macrophages in vascular disease states and on cutting edge genomic tools for the identification and characterization of such targets.

Acute hyperglycaemia induces changes in the transcription levels of 4 major genes in human endothelial cells: macroarrays-based expression analysis.

Hyperglycaemia, in insulin-dependent or independent diabetes mellitus, promotes endothelial cell (EC) dysfunction and is a major factor in the development of macro- or microvascular diseases. The mechanisms and the disease-related genes in vascular diseases resulting from hyperglycaemia are poorly understood. Macroarrays. bearing a total of 588 cDNA known genes, were used to analyze HUVEC gene transcription subjected to 25 or 5-mM glucose for 24 h. Isolated mRNA derived from treated first passage HUVEC were reverse transcribed, 32P labeled, and hybridized to the cDNA macroarrays. Results show that acute hyperglycaemia induces an up-regulation of seven major genes, four of which were not previously reported in the literature. Northern blot analyses, performed on these 4 genes, confirm macroarrays results for alphav, beta4, c-myc, and MUC18. Moreover, time course analysis (0, 2, 4, 8, 2, 16, 24 h) of alphav, beta4 c-myc, and MUC18 mRNA expression, observed by northern blot assays, showed a peak at time points situated between 2 to 8 h. The 3 other genes (ICAM-1, beta1, and IL-8), were shown by others to be significantly upregulated after glucose stimulation. Furthermore, ELISA assays performed on the supernatant of HUVEC culture medium showed a significant increase of IL-8 for cells treated with 25-mM compared to 5-mM glucose. Identified genes, upregulated in endothelial cells as a result of acute hyperglycaemia, may serve as therapeutic or diagnostic targets in vascular lesions present in diabetic patients. These results also demonstrate the use of cDNA macroarrays as an effective approach in identifying genes implicated in a diseased cell.

Effect of a micronized purified flavonoid fraction on in vivo platelet functions in the rat.

The aim of this study was to investigate the effects of a micronized purified flavonoid fraction (MPFF) on in vivo rat platelet functions. Platelet aggregation and disaggregation were evaluated by a noninvasive, automated isotope monitoring system (AimsPlus). Indium-labeled platelets were injected into anesthetized rats and stimulated by adenosine diphosphate (ADP) (10 microg/kg, i.v.) or collagen (50 microg/kg, i.v.). Fibrinogen binding to ex vivo ADP-activated platelets was determined by flow cytometry. MPFF (100 mg/kg, p.o.) significantly reduced ADP-induced platelet aggregation (p<0.05) and increased platelet disaggregation (p<0.05) compared with controls. Moreover, MPFF inhibited collagen-induced platelet aggregation (p<0.001) and increased platelet disaggregation (p<0.01). In addition, fibrinogen binding to 2.5 or 5 microM ADP-stimulated platelets also was reduced significantly (p<0.05 and 0.01, respectively). These results show that MPFF inhibits in vivo rat platelet functions.

Senescent human neutrophil binding to thrombospondin (TSP): evidence for a TSP-independent pathway of phagocytosis by macrophages.

This study investigated the interaction of apoptotic polymorphonuclear neutrophils (PMN) with thrombospondin (TSP), an important event mediating the clearance of apoptotic neutrophils by macrophages. We developed an in vitro assay to examine this interaction. Based on this assay, we found that apoptotic but not fresh PMN bound specifically to surface-immobilized TSP (33 +/- 0.03 x 10(3) cells/well) compared to fibrinogen, fibronectin or laminin (8.0 +/- 0.3 x 10(3) cells/well). Moreover, the binding was specific for surface bound but not soluble TSP and appeared to be divalent cation dependent, was not significantly inhibited by heparin and was sensitive to cycloheximide (CHX) treatment of senescent PMN (>90%) inhibition at 10 microM CHX). In contrast to the binding studies, phagocytosis of senescent PMN by macrophages was not affected by EDTA or cycloheximide. Phosphatidyl-L-serine liposomes, phospho-L-serine, glucosamine, galactosamine, and the acetylated sugars had no effect on phagocytosis. We conclude that: (i) there was specific binding of senescent human PMN to immobilized TSP, which is divalent cation dependent and requires new protein synthesis in the PMN during senescence; (ii) in addition to the recently defined TSP-dependent pathway, there is a TSP-independent pathway mediating phagocytosis of senescent PMN by macrophages. The identity of this pathway remains to be defined.

Identification on human CD36 of a domain (155-183) implicated in binding oxidized low-density lipoproteins (Ox-LDL).

Uptake of oxidized LDL (oxLDL) by macrophages is one of the key events implicated in the initiation and perpetuation of atherosclerotic lesions. One of the major scavenging receptors, which binds modified LDL, on macrophages is CD36. The domain on CD36 implicated in the binding of oxLDL remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA bound FITC-oxidized human LDL in a dose-dependent, saturable manner. This binding was inhibited by an excess of oxLDL but not by native LDL. Anti-CD36 monoclonal antibodies (mAbs) 10/5, FA6-152, and 8A6 (directed against domain 155-183), but not mAb 13/10 (directed against domain 30-76), completely inhibited oxLDL binding to human CD36-transfected COS cells. Cells transfected with a chimeric human CD36 construct (hmh 155-183), resulting from the swapping of human domain 155-183 with its murine counterpart, resulted in low binding of oxLDL. In contrast, cells transfected with a chimeric murine CD36 construct (mhm 155-183), resulting from the swapping of murine domain 155-183 with its human counterpart, resulted in high binding of oxidized human LDL. Binding of oxLDL to cells transfected by chimeric construct mhm 155-183 were only partially blocked by mAbs 10/5, FA6-152, and 8A6. In the present study we have identified, for the first time, an important functional domain (encompassing amino acids 155-183) on CD36 involved in the binding of oxLDL. In addition, the binding site for oxidized human LDL on murine CD36 seems to differ from its human counterpart.

Thrombospondin modulates melanoma--platelet interactions and melanoma tumour cell growth in vivo.

In this study we have investigated the role of thrombospondin (TSP) as a possible ligand playing a key role in human M3Da. melanoma cell interaction with platelets and in tumour growth. TSP is secreted (80 +/- 6 ng TSP 10(-6) cells) and bound to the surface of M3Da. cells via receptors different from CD36, as shown by biosynthetic labelling and immunofluorescence studies. The levels of TSP binding to M3Da. cells evaluated by binding studies, using an anti-TSP monoclonal antibody (MAb) (LYP8), shows 367,000 +/- 58,000 (mean +/- s.d.) LYP8 binding sites per cell with a dissociation constant (Kd) of 67 nM. TSP binding to M3Da. cells shows 400,000 +/- 50,000 TSP binding sites per cell with a Kd of 10 nM. The capacity of anti-TSP MAb (LYP8) to inhibit M3Da.-platelet interactions was followed on an aggregometer and evaluated by electron microscopy studies. The biological role of TSP binding to M3Da. cells was investigated by implanting subcutaneously the M3Da. cell line in nude mice and following the size and time of in vivo tumour growth. Reducing the availability or the functional level of TSP by using an anti-TSP MAb (LYP8) resulted in a significant decrease in platelet aggregates interacting with M3Da. melanoma cells. Using an enzyme-linked immunosorbent assay, purified alpha nu beta 3 was shown to bind TSP. Moreover, LYP8-coated M3Da. cells showed a reduced capacity to form tumours in vivo. M3Da. cells were observed to attach and spread on human platelet TSP-coated plastic wells. This attachment by M3Da. cells was inhibited in a similar way by LYP8 and an anti-alpha nu beta 3 MAb (LYP18). The results obtained in this study show that TSP secreted and bound to the surface of a human melanoma cell line (M3Da.) acts as a link between aggregated platelets and the M3Da. cell surface. Moreover, these results shows that TSP can modulate tumour growth in vivo. Reagents such as MAbs directed against TSP and peptides derived from TSP could not only be used as a new therapeutic approach in the control of tumour metastasis of melanoma, but may also contribute to elucidation of the role of TSP in cancer biology.

A structural/functional domain on human CD36 is involved in the binding of anti-Nak(a) antibodies.

The human CD36 antigen is an integral membrane glycoprotein expressed by platelets, monocytes, endothelial cells and various tumor cell lines. CD36 acts as a receptor for thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein. Individuals possessing the Nak(a)-negative phenotype do not express CD36 and risk developing anti-CD36 isoantibodies upon blood transfusion or during pregnancy. In the present study, we have examined the interaction of an anti-Nak(a) serum with recombinantly expressed CD36. Results obtained show that five functional CD36 monoclonal antibodies (OKM5, FA6-152, L103, ESIV-C7 and 10/5) prevent the binding of the anti-Nak(a) serum whereas a single monoclonal antibody (13/10) has no effect. Consistent with this result, an epitope map of CD36 generated using cross-blocking experiments, indicates that the inhibitory monoclonal antibodies recognize closely-related epitopes whereas 13/10 reacts with a distinct CD36 determinant. Furthermore, we have demonstrated, in a recent study, that OKM5, FA6-152, L103 and 10/5 bind to the same CD36 domain defined by amino acids 155 to 183. Taken together, our results indicate that the 155-183 sequence is important for the binding of the anti-Nak(a) serum to CD36 and may represent a surface-exposed, immunogenic and presumably functional region on human CD36.

Human melanoma cell lines differ in their capacity to release ADP and aggregate platelets.

In this study we have investigated, using three different human melanoma cell lines (M1Do., M3Da., M4Be.). the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (alpha v, beta 3, alpha v beta 3, alpha IIb, alpha v beta 3) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M3Da, as shown by electron microscopy, in contrast to M1Do, which induced a slow reversible aggregation. M4Be. did not induce platelet aggregation. In both cases, with M3Da. or M1Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-alpha v beta 3 monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M1Do., M3Da. and M4Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M3Da., followed by M1Do., and none detected for M4Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.

Two human melanoma cell-line variants with enhanced in vivo tumor growth and metastatic capacity do not express the beta 3 integrin subunit.

The alpha v beta 3 integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express alpha v beta 3. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of alpha v beta 3 expression. Established human melanoma cell lines (M3Da., M4Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, T1P26), derived from a poorly metastatic M4Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the beta 3 integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, T1P26) compared to M3Da. and M4Beu. cell lines which express a relatively high number of beta 3 subunits. The expression levels of the integrin subunits beta 1, beta 5, beta 6 and alpha v were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of beta 3 in 7GP or T1P26 cell lines and its presence in M3Da. and M4Beu. Moreover, similar levels of alpha v transcript were present in the four melanoma cell lines. The functional effect of the absence of beta 3 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and T1P26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M3Da. or M4Beu. cell lines. The beta 3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M4Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of beta 3 or alpha v beta 3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the alpha v beta 3 integrin complex.

Cloning of the cDNA encoding human platelet CD36: comparison to PCR amplified fragments of monocyte, endothelial and HEL cells.

Glycoprotein CD36, also known as GPIIIb or GPIV, is a major platelet glycoprotein that bears the newly identified Naka alloantigen. The aim of this study was to clone platelet CD36 and investigate other forms of CD36-cDNA present in monocytes, endothelial and HEL cells. RNA from above mentioned cells were reverse transcribed (RT), using specific primers for CD36, and amplified by the polymerase chain reaction (PCR) technique. Sequencing the different amplified platelet derived cDNA fragments, spanning the whole coding and flanking regions, showed the near identity between platelet and CD36-placenta cDNA. Platelet CD36-cDNA cross-hybridized, in Southern blots, with RT-PCR amplified cDNA originating from monocytes, endothelial and HEL cells. However, monocytes showed a RT-PCR amplified cDNA fragment (561 bp) that was present in platelets and placenta but not on endothelial on HEL-cells. Northern blot analysis of platelet RNA hybridized with placenta CD36 indicated the presence of a major (1.95 kb) and a minor (0.95 kb) transcript. The 1.95 kb transcript was the only one observed on Northern blots of monocytes, endothelial and HEL cells. These results indicate that the structure of CD36 expressed in platelets is similar, with the exception of the 3' flanking region, to that of placenta. Differences in apparent molecular weight between CD36 and CD36-like glycoproteins may be due to post-translational modifications.

Dacron vascular biomaterial triggers macrophage ectoenzyme activity without change in cell membrane fluidity.

Biomaterials induce an inflammatory reaction characterized by a rapid recruitment at the implantation site of polymorphonuclear cells and macrophages. In the course of the inflammatory response, the cellular activation triggers expression of a number of enzymes, such as 5'-nucleotidase, which is widely distributed in animal cell membranes as an ectoenzyme. It is now well established that 5'-nucleotidase activity decreases following the contact of inflammatory cells with foreign particles. In this paper we investigate a possible correlation between the enzymatic activities and the dynamic properties of the cell membrane bilayer. Dacron pieces were introduced into rats' peritoneal cavities for a period of 6 h, after which the peritoneal cells were harvested, and various enzyme assays performed, including those for cytoplasmic, lysosomal, and ectoenzymes. In parallel, we studied cell membrane fluidity, using fluorescence polarization of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cellular ultrastructural alteration resulting from the cell-biomaterial interactions using scanning and transmission electron microscopy. Our results show that: 1) macrophages spread around the Dacron fibers with cytoplasmic finger-like projections, but no phagolysosomes, 2) 5'-nucleotidase levels decrease with surgical trauma in comparison with the resident cell exudate, 3) implantation of biomaterials slightly modify the 5'-nucleotidase levels observed in the sham animal, 4) no differences in the anisotropy values indicating that membrane lipid order within the cells could not account for the observed decrease of 5'-nucleotidase activity. Thus, we can suggest that 5'-nucleotidase expression may reflect a particular feature of cell activation without a phagocytic process.

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.

The cell adhesion molecule CD31 is phosphorylated after cell activation. Down-regulation of CD31 in activated T lymphocytes.

We report the independent cloning of the cDNA for CD31, a recently described cell adhesion molecule of the immunoglobulin gene superfamily present on platelets, granulocytes, monocytes, lymphocytes, and endothelial cells. Northern analysis revealed three major mRNA transcripts in Jurkat (a human T cell line) and K562 and HEL (leukemia cell lines) cells with an additional 5.3-kilobase transcript seen in cultured human umbilical vein endothelial cells. Following T cell activation, CD31 mRNA was down-regulated by Northern analysis, and decreased CD31 protein expression was confirmed by immunoblots. The down-regulation of CD31 was partially mediated by decreased transcription as demonstrated by nuclear run-on studies. CD31 became rapidly phosphorylated in platelets, Jurkat cells, and endothelial cells after cell activation. We were unable to demonstrate the presence of a phosphotyrosine in CD31 using monoclonal and polyclonal phosphotyrosine antibodies. In addition, CD31 phosphorylation in platelets was induced by phorbol ester and was blocked by staurosporin, a protein kinase C inhibitor, suggesting that CD31 phosphorylation is mediated by protein kinase C and involves serine and/or threonine residues. The phosphorylation of CD31 following cell activation may modulate its cellular adhesiveness, and the down-regulation of its expression may serve to impart target specificity and to localize effector lymphocytes to areas of inflammation.

Separation of important new platelet glycoproteins (GPIa, GPIc, GPIc*, GPIIa and GMP-140) by f.p.l.c. Characterization by monoclonal antibodies and gas-phase sequencing.

A large number of membrane glycoproteins (around 40) are present on the surface of human blood platelets. Some of these glycoproteins are expressed in relatively small amounts, and their functions, as well as their structure, remain to be elucidated. The aim of the present study was to separate rapidly, under non-denaturing conditions, and characterize minor glycoproteins such as Very Late Antigens (VLA) (GPIa, GPIc, GPIc* and GPIIa) and GMP-140 (also known as PADGEM). VLAs and GMP-140 are respectively members of the integrin and selectin families. Platelet membrane glycoproteins were separated by wheat-germ agglutinin lectin affinity and Mono Q anion-exchange f.p.l.c. Peaks bearing isolated glycoproteins were electrophoresed on one- or two-dimensional SDS/polyacrylamide gels, Western blotted on to Immobilon poly(vinylidene difluoride) membranes and gas-phase-sequenced. The identity of isolated glycoproteins was also obtained by the use of monoclonal or polyclonal antibodies and tryptic peptide maps. Five minor [GPIa, GPIc, GPIc*, GPIIa and GMP 140 (PADGEM)], as well as a major (GPIIIb) glycoprotein, were eluted at low salt concentrations. GPIIb-IIIa and GPIb were eluted at high salt concentrations. The N-terminal sequence of platelet GPIa was identical with that obtained by Takada & Hemler [(1989) J. Cell Biol. 109, 397-407]. However, the N-terminal sequence of platelet GPIc + Ic* and GPIIa were found to differ from those deduced from cDNA sequences isolated from human placenta or umbilical-vein endothelial-cell cDNA libraries. The combined use of f.p.l.c. and gas-phase sequencing techniques provides a very powerful tool to separate and characterize rapidly platelet or other cellular proteins for structural, immunological and functional studies.

New families of adhesion molecules play a vital role in platelet functions.

Adhesion molecules play a crucial part in cell-matrix and in cell-cell interactions. These interactions, which are essential to the body's defense processes, involve adhesion molecules belonging to different families: integrins, immunoglobulins and selectins. Integrins are expressed by a large number of tissues, whereas other adhesion molecule families are restricted to a small number of cell types. A recent symposium dealt with the recruitment of circulating platelets at specific sites, their adhesion to extracellular matrix components and their activation by agonists leading to aggregation or attachment to other cells. These events, supporting hemostasis and thrombosis, involve integrins, selectins and other adhesion molecules. This report focuses on newly reported integrins (GPIa, GPIc, GPIIa), selectins (GMP-140) and GPIIIb, previously known as 'minor' surface oriented platelet glycoproteins. Major membrane glycoproteins such as GPIIb-IIIa (an integrin) and GPIb, which also play a vital role in platelet functions, have been extensively reviewed elsewhere.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

Platelet-melanoma cell interaction is mediated by the glycoprotein IIb-IIIa complex.

A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa-like GPs. When the melanoma cells were preincubated with LYP 18, tumor-platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.