Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation

Platelets lysed with Triton X-100 contain 3.44 +/- 1.27 (SD) microgram of fibronectin (cold-insoluble globulin) per 10(9) platelets. Fibronectin was partially released from washed whole platelets by collagen or thrombin, and its release by collagen was inhibited by aspirin. Analysis of subcellular fractions obtained by density-gradient centrifugation of disrupted platelets indicated that fibronectin was contained in the alpha granules. Fibrinogen depleted of fibronectin (less than 2 microgram/mg) supported ADP-induced aggregation as effectively as fibrinogen contaminated with this protein, thus reinforcing the generally held view that fibrinogen itself is the necessary protein cofactor in this reaction.     

Changes in 32P-Content of Phosphatidic Acid and the Phosphoinositides of Rabbit Platelets during Aggregation Induced by Collagen or Thrombin

S ummary . During the ADP-induced shape change and aggregation of washed platelets that have been labelled with ³²PO₄ there is increased incorporation of ³²P into phosphatidic acid (PA) and the phosphoinositides of the platelets. When added to suspensions of washed platelets, collagen and thrombin cause not only a change in shape and aggregation, but also the release reaction. The present experiments were carried out to study the effect of collagen and thrombin on the ³²P-content of PA and the phosphoinositides of washed platelets from rabbits. Both collagen and thrombin caused an increase in the labelling of PA that was apparent when the platelets had changed shape and before aggregation of platelets was detected. Unlike the change in labelling of PA that occurs during ADP-induced aggregation, there was a marked increase in labelling of PA after the commencement of platelet aggregation. Comparison with the maximum response caused by ADP showed that both collagen and thrombin caused a greater increase in the ³²P-content of PA than can be obtained with ADP. Like ADP, collagen and thrombin also caused an increase in labelling of mono-phosphatidylinositol (MPI). Collagen caused an increase in labelling of diphosphatidylinositol (DPI) but not triphosphatidylinositol (TPI). As with ADP in previously reported experiments, the increase in labelling of DPI occurred after the increase in labelling of PA but preceded the increase in labelling of MPI. An increase in the ³²P-content of DPI has not been demonstrated previously in similar in vitro experiments with other cells using other stimuli. The changes in extent of incorporation of ³²P into PA and phosphoinositides may reflect changes in these phospholipids which are important in the mechanism of the change in platelet shape and the release reaction.     

Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood

A parallel-plate perfusion chamber has been used to evaluate the contribution of the adhesive membrane glycoprotein CD36 (GPIV) to platelet adhesion on type I collagen in flowing whole blood at a shear rate of 800 s-1. In one series of experiments, reconstituted normal blood (hematocrit 0.4; platelet count 1.5 x 10(5)/microL) was prepared from washed red blood cells, plasma, and washed platelets that had been incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody (50 micrograms/mL, 30 minutes, 37 degrees C). Percent surface coverage of collagen-coated coverslips using reconstituted blood with antibody-blocked platelets, as compared with paired reconstituted controls (100%), was 50% at 2 minutes, 87% at 5 minutes, and 90% at 10 minutes. Further studies were performed by perfusion of whole blood from a healthy donor of the Naka-negative phenotype, whose platelets constitutively lack CD36, over collagen-coated coverslips. In this case, percent surface coverage was 55% of normal controls at 2 minutes, 76% of controls at 5 minutes, and 72% of controls at 10 minutes. In both preparations, platelets lacking functional CD36 had a statistically significant decrease (P < .005) in adhesion after 2 minutes and 10 minutes perfusion but not at 5 minutes. These results show that functional CD36 facilitates the rapid adhesion of platelets to collagen and that this effect is seen at the earliest time points of their interaction.     

Immunohistological comparison of platelet factor 4 (PF4), fibronectin (Fn) and factor VIII related antigen (VIIIR:Ag) in human platelet granules

S ummary . Immunofluorescence microscopy was used to study the localization of platelet factor 4 (PF4), fibronectin (Fn) and factor VIII related antigen in washed normal platelets and those from patients with severe von Willebrand's disease (vWd). Platelets were prepared by an improved multi-unit modification of the Sayk chamber. This facilitated the preparation of samples with minimum disruption of platelets and readily permitted the demonstration of intact platelet granules by the immunological techniques used. The antigens were demonstrated by treating the same preparations sequentially with appropriate heterologous antisera and species specific fluorescein or rhodamine conjugated antisera. Comparison of the different antigens in identical platelets indicated that Fn and VIIIR:Ag were localized to the same granules as PF4 and the results were thus consistent with their presence in alpha granules. Fn and PF4, but not VIIIR:Ag, were present in platelet granules of patients with severe vWd. The antigens were always detected in the same granules, and major sub-populations of differently stained granules were not observed. The methods were applied to investigate normal platelets aggregated with collagen. Fn and VIIIR:Ag were detected in platelet granules after aggregation although the granules themselves were possibly differently distributed in these samples compared with the non-aggregated platelets. The localization of PF4 could not be reliably assessed in aggregated platelets by these methods. The techniques may be useful in localizing platelet antigens and studying release during aggregation.     

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.     

Impact of fibronectin assembly on platelet thrombus formation in response to type I collagen and von Willebrand factor

Plasma fibronectin enhances platelet thrombus formation on surfaces coated with collagen. We investigated the role of fibronectin assembly in this process. Platelets adherent to fibrillar type I collagen, but not platelets adherent to von Willebrand factor (VWF), supported assembly of plasma fibronectin under static conditions. At a shear rate of 1250 s(-1), platelets adherent to collagen assembled coperfused plasma fibronectin and formed larger thrombi in a fibronectin-concentration-dependent manner, with a maximum effect at 250 mug/mL. Enhanced thrombus formation on collagen was blocked by a peptide that binds to the N-terminal region of fibronectin and inhibits fibronectin assembly. Cross-linking of fibronectin to collagen prior to exposure to platelets had no effect on thrombus formation. Collagen-induced platelet thrombus formation at a shear rate of 5000 s(-1) required coperfusion with VWF and did not result in assembly of coperfused fibronectin. VWF-mediated increase in platelet thrombi on collagen was not enhanced and indeed was somewhat attenuated by coperfused fibronectin at a shear rate of 5000 s(-1). These results indicate that, at moderately high but not very high shear rates, fibronectin assembly in platelet aggregates that form in response to collagen enhances thrombus formation and serves as an alternative to VWF-mediated enhancement.     

Platelet Adherence to Collagen: Role of Plasma, ADP, and Divalent Cations

The effect of varying methods of platelet preparation and the role of ADP and divalent cations in supporting platelet adherence to collagen and the release of 14C-serotonin were assessed by affinity chromatography on collagen/Sepharose to define physiological conditions for this interaction. Platelets were separated by centrifugation or gel filtration from blood anticoagulated with EDTA or citrate and suspended in native plasma or buffer. After labelling with 51Cr or 14C-serotonin, they were passed though columns of collagen covalently linked to cyanogen bromide-activated Sepharose. Adherence to collagen was less in plasma as compared to buffer, was increased by centrifuging the platelets before testing, and was unaltered by addition of ADP. Removal of ADP with CP/CPK decreased the adherence of gel-filtered citrated and EDTA platelets and washed EDTA platelets (P less than 0.001) but not EDTA platelets in plasma. Adherence and release of citrated platelets in plasma or buffer containing CP/CPK were greater than that of EDTA platelets (P less than 0.01); no difference existed with gel-filtered platelets. The addition of 1 mM Mg++ to citrate or EDTA-anticoagulated washed platelets increased adherence and release (P less than 0.01). The results indicate that the choice of experimental conditions affect the assessment of factors which influence or promote platelet interaction with collagen. Platelet-collagen adherence is enhanced by laboratory manipulations and partially inhibited by normal plasma. Maximal adherence and release occur when divalent cations, particularly Mg++, and ADP are available. Their absence reduces but does not inhibit these reactions.     

Platelet stimulation by antifibronectin antibodies requires the Fc region of antibody.

Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody with platelet membrane fibronectin was responsible for this stimulation, we prepared proteolytic fragments of the antifibronectin antibodies. Purified F(ab')2 fragments had a greatly diminished ability to elicit degranulation, and rabbit F(ab')2 fragments were totally ineffective in this regard. Preimmune IgG from these sources was capable of inhibiting antibody-induced serotonin release in a dose-dependent manner. Purified antibody Fc fragments also inhibited this stimulation in doses stoichiometrically equivalent to the inhibition seen with preimmune IgG. These results suggest that (i) platelet stimulation elicited by anti-human fibronectin antibody is mediated by the platelet Fc receptor and therefore is likely to be dependent on antigen-antibody complex formation and (ii) fibronectin on the platelet surface may not be the primary receptor for platelet adherence to collagen.     

Organizing Committee:

Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37°C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37°C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.     

The role of fibronectin in platelet aggregation

A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.     

Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.     

Thrombin increases the adhesion of washed human platelets to collagen

Thrombin stimulates the adhesion of washed human platelets to fibrillar collagen. This phenomenon occurs also when platelets, before thrombin stimulation, are resuspended in the presence of prostaglandin E1 to minimize the release reaction. Enzymatic activity of thrombin is not necessary for the enhancement of platelet adhesiveness, since phenylmethylsulphonylfluoride inhibited thrombin is effective in this respect. Detachment of thrombin from thrombin treated platelets by the use of hirudin restores normal platelet adhesiveness to collagen.     

The metalloprotease,NN-PF3 from <Emphasis Type="Italic">Naja naja</Emphasis> venom inhibits platelet aggregation primarily by affecting α2β1 integrin

NN-PF3 is a non-toxic, anticoagulant, high-molecular-mass (67.81 kDa) metalloprotease from Indian cobra (Naja naja) venom. In the present study, NN-PF3 was investigated for the mechanism of inhibition of collagen-induced aggregation of human platelets. The complete inhibition of collagen-induced aggregation and partial inhibition of ADP- and epinephrine-induced aggregation has the respective IC₅₀ of 75 ± 5, 185 ± 10, and 232 ± 12 nM, whereas no inhibition of thrombin-, arachidonic acid-, and ristocetin-induced aggregation of platelets was observed in platelet-rich plasma. Further, native NN-PF3 and EDTA-inactivated NN-PF3 inhibited collagen-induced aggregation of washed platelets with respective IC₅₀ of 75 ± 4 and 180 ± 6 nM. The higher inhibitory effect of native NN-PF3 compared with EDTA-inactivated NN-PF3 suggests the enzymatic and non-enzymatic mechanism of inhibition. NN-PF3 pretreatment affected the collagen binding but not the fibrinogen, and fibronectin binding of washed platelets in adhesion assay suggested that the collagen receptors are affected. Western blot study using anti-integrin α2β1 mAb 6F1 suggested that NN-PF3 binds to integrin α2β1 in a primary structure-dependent manner only and is not cleaved. There was a drastic reduction in the intensity of several intracellular signaling phosphotyrosine protein bands when monoclonal anti-phosphotyrosine antibody was used, suggesting that the major activation pathway of platelets get affected, which occurs through glycoprotein VI. NN-PF3 did not bind to collagen as revealed by Western blot using anti-collagen mAb. Furthermore, neither the proteolytic cleavage of fibrinogen nor its degradation products by NN-PF3 contributed for the collagen-induced platelet aggregation inhibition.     

Hydrogen peroxide as trigger of platelet aggregation.

In order to verify if H2O2 affects platelet function, platelet-rich plasma and human washed platelets were incubated with subthreshold concentrations (STC) of collagen or arachidonic acid or ADP and/or with 75-150 microM H2O2. While H2O2 alone did not affect platelet aggregation, it amplified platelet aggregation response in samples stimulated with STC of arachidonic acid and collagen but not in samples stimulated with STC of ADP. When platelets were preventively treated with aspirin, a cyclooxygenase inhibitor, the platelet activation by H2O2 was not observed. Thromboxane A2 (TxA2) was not produced by human washed platelets stimulated with STC of arachidonic acid, collagen or by H2O2 alone. On the contrary, when STC of agonists were tested on platelets supplemented with H2O2 an evident TxA2 production was seen. This effect was prevented by aspirin pretreatment or by the addition of catalase, an enzyme which destroys H2O2. This study suggests that H2O2 triggers the activation of platelets exposed to STC of collagen and arachidonic acid, via the cyclooxygenase pathway.     

Variability of integrin alpha 2 beta 1 activity on human platelets

The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

Fibronectin and the multiple interaction model for platelet-collagen adhesion.

A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.     

The inhibitory effects of exogenous arachidonic acid on rabbit platelet aggregation and the release reaction

Although arachidonic acid causes rabbit platelet aggregation and the release of granule contents in suspensions of washed platelets when used in concentrations of approximately 50-300 microM, higher concentrations (500 microM) cause neither aggregation nor release. Suspensions of platelets from rabbits wee exposed to arachidonic acid (250 microM) for 15 min, allowed to recover in the presence of PGE1 for 30 min, washed, and resuspended; in some experiments, the platelets were treated with aspirin before being exposed to arachidonic acid. Aggregation of platelets pretreated with arachidonic acid was inhibited in response to ADP; this effect was greater with the non-aspirin- treated platelets and persisted for at least 4 hr after resuspension. The association of 125I-fibrinogen with the platelets as a result of ADP stimulation was also inhibited. Aggregation and release of granule contents in response to collagen and low concentrations of thrombin was inhibited, but the inhibition could be overcome by higher concentrations. Thrombin induced further release of granule contents from platelets exposed to arachidonic acid without pretreatment with aspirin. Platelets that had been exposed to arachidonic acid, either with or without pretreatment with aspirin, did not aggregate or undergo further release upon stimulation with arachidonic acid after they were washed and resuspended. Inhibition of the lipoxygenase pathway with eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) did not affect the inhibition caused by arachidonic acid, so it is unlikely that a product of this pathway is responsible for the inhibition. Mixing experiments indicated that the pretreated platelets did not form a thromboxane-A2-like activity, and that they were unresponsive to aggregation and release induced by products formed from arachidonic acid. Experiments with 3H-arachidonic acid showed that after 45 min of incubation with platelets, only 1.1% of the 3H-arachidonic acid remained as free arachidonic acid in the platelets. Although cyclic-AMP was slightly increased 1 min after the addition of arachidonic acid, the cyclic-AMP concentration was the same as that of control platelets after the platelets were washed and resuspended, indicating that increased cyclic-AMP is not likely to be responsible for the persistent inhibitory effect. Thus, the inhibitory effect of pretreatment with arachidonic acid is a general effect on responses to a variety of aggregating agents that act through different mechanisms, and the inhibition is not related to thromboxane-A2 formation. The possibility of membrane perturbation resulting in the unavailability of receptors may explain the persistent inhibitory effect, but the responsible reactions have not been identified.     

Lebecetin, a C-lectin protein from the venom of Macrovipera lebetina that inhibits platelet aggregation and adhesion of cancerous cells

A novel C-lectin protein, lebecetin, was purified and characterized from the venom of Macrovipera lebetina. It is a disulfide-linked heterodimer of 15 and 16 kD. The subunits are homologous to each other and to the other snake venom proteins of the C-type (Ca(2+)-dependent) lectin superfamily. Lebecetin shows a potent inhibitory effect on whole blood and washed platelets induced by different agonists. It inhibits the agglutination of human fixed platelets in the presence of ristocetin. Lebecetin also interferes with the adhesion of IGR39 melanoma and HT29D4 adenocarcinoma cells. These two lines adhere to lebecetin used as matrix. Lebecetin is also able to strongly reduce IGR39 and HT29D4 cell adhesion to fibrinogen and laminin, but not to fibronectin and collagen types I and IV, respectively. Adhesion properties of lebecetin may thus involve integrin receptors.     

Evidence that fibronectin is the collagen receptor on platelet membranes.

Evidence is presented that fibronectin is present on the platelet cell membrane and that it is a receptor for collagen in the platelet-collagen interaction. First, sodium dodecyl sulfate/acrylamide gel electrophoresis was performed on the proteins remaining attached to the surface of collagen after the removal of the remainder of the platelet by sonication. The material was relatively enriched in a glycoprotein that comigrated with cold-insoluble globulin (CIG), a form of fibronectin, and in other proteins which comigrated with myosin, actin, and tropomyosin. The presumptive presence of contractile proteins is consistent with the presence of microfibrillar proteins. Second, the collagen-attached material was shown to contain a protein that reacted with anti-CIG serum by immunoelectrophoresis. Third, when CIG was preincubated with fibrous collagen, the platelet-collagen interaction was inhibited. Fourth, rabbit anti-human CIG stimulated human platelets to secrete the contents of their dense granules. The stimulation was not due to antibody complexes present in the solution. Fifth, a protein was extracted from well-washed platelets and purified on affinity columns of anti-CIG antibodies. The isolated protein was found to bind to fibrous collagen.