Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.     

Activation of platelet heparitinase by tumor cell-derived factors

The nature of the cooperation between platelets and tumor cells during the process of blood-borne metastasis is essentially unknown. In previous in vitro studies we showed that platelets participated in the formation of gaps in the endothelial cell lining, and that concomitantly heparan sulfate glycosaminoglycans were degraded by the platelet heparitinase, released on activation of platelets. In the current study we show that the ability to degrade proteoheparan sulfate derived from endothelial extracellular matrix is gradually eliminated when the number of human platelets is decreased from 5 x 10(7) to 10(6) cells/mL. When aliquots of conditioned media or lysates of either Eb or heat-inactivated ESb mouse lymphoma cells (both of which showed no heparanase activity) were added to freeze-thawed lysates of 10(6) platelets, a reappearance of platelet heparitinase activity was observed. A similar activation was not elicited by lysates of several normal mammalian cells. These data suggest that in its native form, a fraction of the platelet heparitinase is stored in an inactive form that can be activated by a factor secreted by lymphoma, but not by normal cells. Partial characterization of the heparitinase-activating factor showed that it is a heat-stable polyanionic molecule, devoid of proteolytic activity and resistant to both proteolytic and chondroitinase digestions. Activation of platelet heparitinase was also observed on coincubation with chondroitinases ABC and AC, suggesting that the inactive form of platelet heparitinase could result from a complex formation with a chondroitinase-sensitive proteoglycan. The lymphoma-derived heparitinase activating factor itself is, however, not a chondroitinase, because activity of chondroitinase could not be detected in Eb and ESb cells. A possible mechanism by which tumor cells recruit and regulate the activity of platelet heparitinase, and its relevance to the progression of blood borne metastasis, is discussed.     

Degranulating mast cells secrete an endoglycosidase that degrades heparan sulfate in subendothelial extracellular matrix.

Mast cells are widely distributed in perivascular connective tissues, especially in areas of active tumor growth and vascular reactivity. Incubation of metabolically [35S]O4 = -labeled subendothelial extracellular matrix (ECM) with lysates of bone marrow-derived mouse mast cells (BMMC) resulted in extensive degradation of heparan sulfate (HS) into fragments 5 to 6 times smaller than intact HS side chains. A much lower activity (seven- to eightfold) was expressed by intact BMMC incubated in contact with the ECM. These fragments were not produced in the presence of heparin, were sensitive to deamination with nitrous acid, and resistant to further degradation with papain or chondroitinase ABC. These results indicate that an endoglycosidase (heparanase) is involved in BMMC-mediated degradation of HS in the subendothelial ECM. Heparanase activity was not detected in medium conditioned by cultured BMMC, or in lysates of Ableson transformed BMMC and rat basophilic leukemic (RBL) cells. Both heparanase and beta-hexosaminidase, a mast cell granule enzyme, were released on degranulation of BMMC induced by the calcium ionophore A23187, or by exposure to IgE-Ag, suggesting that heparanase is localized in the cell granules. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase were released. Degradation of the ECM-HS by the mast cell heparanase and the associated release of HS-bound endothelial cell growth factors that are stored in ECM (Vlodavsky et al, Proc Natl Acad Sci USA 84:2292, 1987; Bashkin et al, Biochemistry 28:1737, 1989) may play a role in the proposed mast cell-mediated stimulation of neovascularization.     

Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin''s B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, releasing saccharide products with appreciable size (4–7 kDa) and biological potency. Enzymatic degradation of HS leads to disassembly of the extracellular matrix (ECM) and correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the ECM and basement membrane underlying epithelial and endothelial cells (1, 2). Heparanase expression is induced in human cancer, most often associating with reduced patients’ survival postoperation, increased tumor metastasis, and higher vessel density (3–5). In addition, heparanase up-regulation is associated with tumors larger in size (3, 5). Likewise, heparanase over-expression enhanced (6, 7), whereas local delivery of anti-heparanase siRNA inhibited (8), the growth of tumor xenografts. These results imply that heparanase function is not limited to tumor metastasis but is engaged in progression of the primary lesion, thus critically supporting the intimate involvement of heparanase in tumor progression and encouraging the development of heparanase inhibitors as anticancer therapeutics (9–12). As a consequence, heparanase inhibitors are currently evaluated in phase 1 clinical trials (13).Heparanase activity is similarly implicated in the progression of multiple myeloma (14–16), but its significance in other hematologic malignancies has not yet been characterized. Lymphomas are a heterogeneous group of cancers that arise from developing lymphocytes and produce tumors predominantly in lymphoid structures (i.e., bone marrow), but also in extranodal tissues. Collectively, lymphomas constitute the fifth most common cancer in North America, with more than 90% of the patients being affected by lymphomas of B-cell origin (17). Despite overall improvements in outcomes of lymphoma, ∼30–40% of patients have disease that is either refractory or relapses after standard therapy (18). Therefore, a better understanding of the molecular pathobiology of lymphomas is needed for the development of new therapeutic approaches. Here, we provide evidence that heparanase is expressed by B-lymphomas and that heparanase inhibitors restrain tumor growth. Furthermore, we describe the development of novel heparanase-neutralizing monoclonal antibodies (mAbs) that attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.     

Morphological response of blood platelets to increased venular permeability in vivo

Electron microscopy is used to investigate the response of blood platelets to isoproterenol and paracentesis-induced changes in the morphology and permeability of iridial venules in young (3–5 weeks) and older (6–8 months) rats. In isoproterenol-treated eyes of young and older rats, a notable response of the venular endothelium occurred at 1–5 min. Small patent gaps were occasionally seen between adjacent endothelial cells and numerous adlumenal protrusions and membranous vesicles of the endothelial cell were present. Individual platelets or small aggregates were closely associated with the adlumenal protrusions and membranous vesicles. Images suggestive of degranulation were seen in platelets from young animals. At 20 min, alterations of the endothelium were less prominent. In young rats, individual platelets were often surrounded by clusters of carbon particles, but were not adherent. In older rats, there was a marked adhesion of individual platelets to the endothelium in the immediate vicinity of patent gaps. Clusters of carbon particles were adhering to the platelet and dense alpha granules were characteristically present. At 2 hr, the endothelium appeared normal but in the older animal only, platelets were still adherent and associated with carbon particles. In the paracentesis experiments, patent gaps and adlumental protrusions and membranous vesicles of endothelial cells were again observed. In young rats at 20 min, small gaps were occasionally seen, and adlumenal modifications prominent. Clusters of a few platelets (less than six) were present. Within clusters platelets rarely came into close contact with one another, and possessed long pseudopods. An occasional platelet was closely associated with protrusions of the endothelium. At 1 hr the platelet response had subsided. Platelets were less numerous, and pseudopods less prominent. In older rats at 20 min, patent gaps were large and numerous. Large clusters of platelets with well-developed pseudopods were present and closely associated with carbon particles, fibrin, and a dense amorphous precipitate. Individual platelets with pseudopods were present within gaps. Degranulation was not observed. At 2–2.5 hr the endothelium had recovered. Degranulated platelets were seen adhering to the endothelium. The ultrastructural response of platelets described above, differs from that described by others in experiments designed to characterize in vivo changes in the morphology of platelets in the microcirculation. The significance of these results is discussed.     

肝素、低分子肝素的抗肿瘤作用研究进展

肝素、低分子肝素是临床常用的一种抗凝药物.近年来研究发现,其还具有多种生物活性和临床用途,包括抗血管生成及抗肿瘤作用等.肝素、低分子肝素可通过改变肿瘤细胞的细胞和分子学环境而干预肿瘤的发展,例如,肝素、低分子肝素具有抗肿瘤细胞增殖作用,抑制血管内皮细胞增殖和抑制血管形成,通过抑制乙酰肝素酶、基质金属蛋白酶等酶的活性及作用阻止细胞外基质及基底膜的降解,抑制肿瘤细胞黏附及其随后向组织的侵袭、迁移等.     

肝素、低分子肝素的抗肿瘤作用研究进展

肝素、低分子肝素是临床常用的一种抗凝药物.近年来研究发现,其还具有多种生物活性和临床用途,包括抗血管生成及抗肿瘤作用等.肝素、低分子肝素可通过改变肿瘤细胞的细胞和分子学环境而干预肿瘤的发展,例如,肝素、低分子肝素具有抗肿瘤细胞增殖作用,抑制血管内皮细胞增殖和抑制血管形成,通过抑制乙酰肝素酶、基质金属蛋白酶等酶的活性及作...     

Platelet-dependent primary hemostasis promotes selectin- and integrin- mediated neutrophil adhesion to damaged endothelium under flow conditions

Co-localization of blood platelets and granulocytes at sites of hemostasis and inflammation has triggered an intense interest in possible interactions between these cellular processes and induction of vessel wall injury. Leukocyte adhesion to endothelial cells decreases with increasing shear and is dependent on an initial rolling phase mediated by selectins. We hypothesized that flow-dependent platelet adhesion at an injured vessel wall will lead to P-selectin expression by platelets, thus mediating leukocyte co-localization. A perfusion chamber was used in which flowing whole blood induced platelet adhesion to a subendothelial matrix (ECM) of cultured human umbilical vein endothelial cells (HUVEC). We compared neutrophil (polymorphonuclear leukocyte [PMN]) interactions with HUVEC and their ECM with and without adhered platelets. PMNs adhered predominantly to ECM-adhered platelets and not to endothelial cells. ECM alone did not support PMN adhesion under flow conditions. PMN adhesion to unstimulated HUVEC was only substantial at low shear (up to 200 cells/mm2 at shear stress 80 mPa). In marked contrast, PMN adhesion to ECM-adhered platelets was dramatically increased, and adhesion was demonstrated at much higher shear stress (up to 640 mPa). Studies with specific antibodies showed that the platelet-dependent neutrophil adhesion was selectin-mediated. Inhibition of P-selectin caused a marked inhibition of adhesion at high shear stress, whereas the role of leukocyte L-selectin was less pronounced. beta2-Integrin-blocking antibodies inhibited static neutrophil adhesion. fMLP induced L-selectin shedding from leukocytes, resulting in decreased leukocyte adhesion. In conclusion, platelet- dependent hemostasis at the ECM appears to be a powerful intermediate in neutrophil-vessel wall interactions at shear stresses that normally do not allow neutrophil adhesion to intact endothelium.     

Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow

Resting platelet adhesion to inflammatory vascular endothelium is thought to play a causal role in secondary thrombus formation or microcirculatory disturbance after vessel occlusion. However, though adhesion receptors involved in platelet-matrix interactions have been extensively studied, the molecular mechanisms involved in platelet-endothelium interactions are incompletely characterized and have been mainly studied under static conditions. Using human platelets or platelets from wild-type and CD47-/- mice in whole blood, we demonstrated that at low shear rate, CD47 expressed on human and mouse platelets significantly contributes to platelet adhesion on tumor necrosis factor-alpha (TNF-alpha)-stimulated vascular endothelial cells. Using the CD47 agonist peptide 4N1K and blocking monoclonal antibodies (mAbs), we showed that CD47 binds the cell-binding domain (CBD) of endothelial thrombospondin-1 (TSP-1), inducing activation of the platelet alphaIIbbeta3 integrin that in turn becomes able to link the endothelial receptors intercellular adhesion molecule 1 (ICAM-1) and alphavbeta3. Platelet CD36 and GPIbalpha are also involved because platelet incubation with blocking mAbs directed against each of these 2 receptors significantly decreased platelet arrest. Given that anti-CD47 treatment of platelets did not further decrease the adhesion of anti-CD36-treated platelets and CD36 is a TSP-1 receptor, it appears that CD36/TSP-1 interaction could trigger the CD47-dependent pathway. Overall, CD47 antagonists may be potentially useful to inhibit platelet adhesion on inflamed endothelium.     

Interaction of platelets and blood vessels--vascular injuries induced by platelet activation in vivo.

We have found that vascular injuries are induced by intravascular aggregation of platelets activated by arachidonic acid (AA) or ADP. The characteristic findings are the appearance of vacuoles in endothelial cells and eventual deendothelialization. In deendothelialized regions, formation of platelet thrombi was observed. The platelets in the thrombi were stained with 2T60, a monoclonal antibody that recognizes activated platelets. The change was more remarkable in the AA-injected animals because AA has a stronger platelet activating effect and a detergent effect on the endothelium. The ADP-injection experiments clarified the role of platelets in vascular injury. These findings suggest that activated platelets play a role in the genesis of vascular injuries, and that their role is related to thrombus formation.     

Activated platelets mediate Staphylococcus aureus deposition on subendothelial extracellular matrix: the role of glycoprotein Ib

Extracellular matrix (ECM) derived from bovine corneal endothelial cells was used as a model to study the role of platelets in Staphylococcus aureus interaction with the vascular subendothelium. In whole blood, S. aureus activated platelets, as demonstrated by P-selectin expression on the platelet membrane. Subjecting platelet-rich plasma (PRP) to the ECM under oscillatory conditions resulted in platelet adhesion and aggregation. S. aureus increased platelet deposition on ECM depending on the bacterium-platelet ratio. Scanning electron microscopy revealed that S. aureus adhered to and formed clusters on ECM-bound platelets. These findings were confirmed by using [3H]thymidine-labeled bacteria that adhered to the surface more extensively after deposition of platelets on ECM. Platelet pre-treatment with prostaglandin E1 resulted in inhibition of bacterial adherence. Glycoprotein (GP)Ib was involved in the bacterium-platelet interaction, as indicated by the following: (i) S. aureus diminished the binding of GPIb but not of GPIX or GPIIb-IIIa monoclonal antibodies (Mab) to washed fixed platelets; (ii) GPIb Mab inhibited S. aureus -induced platelet aggregation in a dose-dependent fashion; (iii) blockade of von Willebrand factor (vWf) binding to GPIb by a recombinant vWf fragment reversed the enhanced platelet deposition on ECM in the presence of S. aureus but did not affect platelet deposition in the absence of bacteria. The results indicate that S. aureus activates platelets and promotes their deposition on ECM via GPIb-dependent mechanism and that adherent platelets mediate S. aureus deposition on the subendothelium. These interactions might play a role in the pathogenesis of bacterial endocarditis.     

Activated platelets mediate Staphylococcus aureus deposition on subendothelial extracellular matrix: the role of glycoprotein Ib

Extracellular matrix (ECM) derived from bovine corneal endothelial cells was used as a model to study the role of platelets in Staphylococcus aureus interaction with the vascular subendothelium. In whole blood, S. aureus activated platelets, as demonstrated by P-selectin expression on the platelet membrane. Subjecting platelet-rich plasma (PRP) to the ECM under oscillatory conditions resulted in platelet adhesion and aggregation. S. aureus increased platelet deposition on ECM depending on the bacterium-platelet ratio. Scanning electron microscopy revealed that S. aureus adhered to and formed clusters on ECM-bound platelets. These findings were confirmed by using [3H]thymidine-labeled bacteria that adhered to the surface more extensively after deposition of platelets on ECM. Platelet pre-treatment with prostaglandin E1 resulted in inhibition of bacterial adherence. Glycoprotein (GP)Ib was involved in the bacterium-platelet interaction, as indicated by the following: (i) S. aureus diminished the binding of GPIb but not of GPIX or GPIIb-IIIa monoclonal antibodies (Mab) to washed fixed platelets; (ii) GPIb Mab inhibited S. aureus -induced platelet aggregation in a dose-dependent fashion; (iii) blockade of von Willebrand factor (vWf) binding to GPIb by a recombinant vWf fragment reversed the enhanced platelet deposition on ECM in the presence of S. aureus but did not affect platelet deposition in the absence of bacteria. The results indicate that S. aureus activates platelets and promotes their deposition on ECM via GPIb-dependent mechanism and that adherent platelets mediate S. aureus deposition on the subendothelium. These interactions might play a role in the pathogenesis of bacterial endocarditis.     

Endothelium-derived relaxing factor reduces platelet adhesion to bovine endothelial cells.

The adhesion of thrombin-stimulated human blood platelets to either the endothelial surface of intact bovine aorta or cultured bovine aortic endothelial cells was studied to determine the role of endothelium-derived relaxing factor in the regulation of platelet adhesion. Endothelial cells and platelets were pretreated with indomethacin to prevent the formation of prostaglandins. The adhesion of thrombin-stimulated platelets to endothelial cells was reduced by superoxide dismutase and bradykinin. The inhibitory effect of both drugs was abolished by hemoglobin and was absent in strips of bovine aorta where the endothelial cells had been removed by scraping. It is suggested that the effects of bradykinin are mediated by the release of endothelium-derived relaxing factor, which is protected from destruction by superoxide dismutase, and that endothelium-derived relaxing factor contributes to the nonadhesive properties of the vascular endothelium.     

Platelet-derived growth factors

Blood platelets are a rich source of growth factors, including platelet-derived growth factor, platelet-derived endothelial cell growth factor, and transforming growth factor beta. Platelet-derived growth factor stimulates the growth of mesenchymal cells such as fibroblasts and vascular smooth muscle cells, whereas platelet-derived endothelial cell growth factor is a mitogen for vascular endothelial cells. Transforming growth factor beta is a bifunctional regulator of cellular growth, but acts as a potent inhibitor for most cell types. Most of the growth regulatory substances in platelets have been reported to reside in platelet alpha-granules, but platelet-derived endothelial cell growth factor appears to be present in platelet cytoplasm. These growth factors may act at sites of injury as wound hormones. Moreover, they play important roles for some pathological conditions such as atherosclerosis, myelofibrosis, connective tissue diseases, and neoplastic disorders.     

The discovery of nitric oxide as the endogenous nitrovasodilator

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent released by vascular endothelium that mediates the relaxation induced by some vasodilators, including acetylcholine and bradykinin. EDRF also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to vascular endothelium. These actions of EDRF are mediated through stimulation of the soluble guanylate cyclase and the consequent elevation of cyclic guanosine 3',5'-monophosphate. EDRF has been identified as nitric oxide (NO). The pharmacology of NO and EDRF is indistinguishable; furthermore, sufficient NO is released from endothelial cells to account for the biological activities of EDRF. Organic nitrates exert their vasodilator activity following conversion to NO in vascular smooth muscle cells. Thus, NO may be considered the endogenous nitrovasodilator. NO is synthesized by vascular endothelium from the terminal guanido nitrogen atom(s) of the amino acid L-arginine. This indicates the existence of an enzymic pathway in which L-arginine is the endogenous precursor for the synthesis of NO. The discovery of the release of NO by vascular endothelial cells, the biosynthetic pathway leading to its generation, and its interaction with other vasoactive substances opens up new avenues for research into the physiology and pathophysiology of the vessel wall.     

Effect of intact endothelium against platelet-induced coronary artery spasm in isolated rabbit hearts

Effects of collagen-activated washed rabbit platelets on coronary arteries with and without intact endothelium were studied in a supported rabbit heart preparation using a perfluorocarbon (FC-43) as perfusate. The vascular diameter of obtuse marginal coronary arteries was determined by means of gated color arteriography (injection of patent blue dye). Endothelial denudation of the obtuse marginal artery was accomplished by scraping the lumen with a roughened plastic tubing. Injection of washed platelets (10 ml with about 500,000 platelets/μl) not treated with collagen failed to constrict coronary arteries either with intact or denuded endothelium. In contrast, injection of platelet suspension immediately after activation with collagen caused vasoconstriction of denuded obtuse marginal coronary arteries In 10 of 14 cases. In 6 preparations occlusion was complete, lasting up to 16 minutes. In arteries with intact endothelium, no coronary vasoconstriction occurred. In hearts with coronary artery spasm, total coronary vascular resistance increased significantly. This study furnishes additional evidence that endothelial lesions are a contributory factor for large coronary artery spasm and that endothelial cells possess a protective function against vasoconstrictor substances released from aggregating platelets.     

Potential role of platelets in the pathogenesis of tumor metastasis

Platelet activity may be involved in tumor metastasis. The tumor cells, after detachment from the primary site, adhere to vascular endothelium at distant sites and proliferate. Platelets form aggregates with tumor cells in circulation, facilitating their adhesion to the vascular endothelium. Formation of platelet-tumor cell aggregates and their sequestration in various end-organs may result in thrombocytopenia. Certain tumor cell lines directly stimulate platelet activity, some by releasing platelet-aggregating material, a urea-extractable membrane component, others by release of cathepsin, and still others by undefined mechanisms. The direct effect of platelets on tumor cells may be of pathogenic significance. For example, platelet-derived factors stimulate growth of some tumors, whereas others increase vascular permeability and thus facilitate migration of tumor cells across the vessel wall. Lack of these platelet factors, as in thrombocytopenic animals, may indeed inhibit tumor metastasis. Arachidonic acid metabolism in platelets and the vessel walls may contribute to metastatic process. In particular, thromboxane A2 and prostacyclin generation capabilities appear to be important in modulating platelet- tumor cell deposition and growth. To alter the metastatic process, several preliminary trials of platelet-inhibitory agents have been performed. However, the results of these trials have been equivocal, perhaps related to nonspecific effects of these agents on arachidonic acid metabolism. Studies directed at specific pathways of platelet- vessel wall interaction on some tumors appear promising. These newer agents may be of therapeutic value in man.     

Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intercellular collagen matrix.

Human umbilical endothelial cells (ECs) were grown on fibrillar type I collagen in 16.4-mm multiwell tissue culture plates. Human platelets were added to the wells, and platelet adhesion to collagen was examined by scanning electron microscopy and radioisotopic technique in the absence of ECs and in preconfluent and confluent EC cultures. Single adherent platelets of different shapes as well as small aggregates were seen on collagen surface. Human plasma fibronectin added to the system stimulated platelet adhesion and their spreading on collagen. ECs had no effect on the percentage of platelets adherent to collagen-coated gaps in preconfluent culture but decreased the number of spread platelets. It is demonstrated that collagen-coated gaps can bind 14C-labeled liposome--antibody and 14-C-labeled liposome--fibronectin conjugates. ECs grown on fibrillar collagen are suggested as useful models for screening of antiplatelet drugs and for the study of drug targeting to the areas of vascular injury for prevention of thrombosis.     

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cell-induced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (Abciximab) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. Abciximab significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by Abciximab (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab')(2) reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.