Platelet adhesion to collagen and endothelial cell matrix under flow conditions is not dependent on platelet glycoprotein IV

Platelet membrane glycoprotein IV (GPIV) is a cell-surface glycoprotein that has been proposed as a receptor for collagen. Recently, it has been shown that platelets with the Naka-negative phenotype lack GPIV on their surface, whereas donors with this phenotype are healthy and do not suffer from hematologic disorders. In this study, we compared Naka- negative platelets with normal platelets in adhesion to collagen types I, III, IV, and V and the extracellular matrix of endothelial cells (ECM) under static and flow conditions. No differences in platelet adhesion and subsequent aggregate formation on the collagens types I, III, and IV were observed under static and flow conditions. Adhesion of both homozygous and heterozygous Naka-negative platelets to collagen type V was strongly reduced under static conditions. Collagen type V was not adhesive under flow conditions. No difference in platelet adhesion to ECM was observed, which suggests that GPIV is not important in adhesion to subendothelium, for which ECM may serve as a model. These results indicate that GPIV is not a functional receptor for collagen under flow conditions.     

Mediation of platelet adhesion to fibrillar collagen in flowing blood by a proteolytic fragment of human von Willebrand factor

The effect of purified von Willebrand Factor (vWF) fragments, SpII (dimer of two 110 kd subunits) and SpIII (dimer of two 170 kd subunits) obtained with S aureus V-8 protease was tested upon platelet adhesion to collagen. Purified fibrillar human collagen coated onto cover slips was incubated with SpII, SpIII, or undigested vWF and exposed to reconstituted human blood in a parallel-plate perfusion chamber at a high shear rate. Platelet-collagen interactions were estimated using 51Cr-platelets and quantitative morphometry. When blood was reconstituted with citrated autologous plasma, SpIII and vWF strikingly enhanced platelet adhesion to collagen whereas SpII had no effect. When blood was reconstituted with human albumin and divalent cations, SpIII and vWF again promoted platelet adhesion to collagen. In conclusion, our data suggest that (1) SpIII, the N-terminal portion of vWF which binds to platelet membrane glycoprotein Ib, functionally substitutes for vWF in supporting platelet adhesion to collagen; (2) SpII, the C- terminal portion which binds to glycoprotein IIb/IIIa, has no such effect; (3) in addition to its platelet binding domain, SpIII contains another site for binding to collagen; and (4) the multimeric structure of vWF is not required for platelet adhesion to collagen.     

Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen.

Fibronectin mediates the adhesion of cells to collagen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectin-mediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GD1a were the most effective inhibitors followed by GD1b greater than GM1 greater than GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GD1a modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin.     

Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow.

We have used real-time video microscopy to study the mechanisms of platelet adhesion to type I collagen fibrils of distinct structure exposed to flowing blood. Electron microscopy analysis by surface replication demonstrated morphological differences between acid-insoluble fibrils, displaying a regularly repeating striated pattern (banded collagen), and acid-soluble fibrils generated by pepsin treatment of insoluble collagen, smaller in size with a helical configuration (nonbanded collagen). These structural differences proved to be related to the role of platelet integrin alpha(2)beta(1) in stabilizing adhesion to collagen under a variety of flow conditions. Blocking alpha(2)beta(1) function with a monoclonal antibody had no effect on platelet adhesion to insoluble type I collagen coated at high density on a glass surface, whereas there was an absolute dependence of alpha(2)beta(1) function for the initial permanent arrest of platelets and subsequent thrombus formation on pepsin-solubilized type I collagen under the same conditions. In contrast, reconstituted, banded fibrils prepared from pepsin-solubilized type I collagen supported platelet adhesion and thrombus development even when platelet alpha(2)beta(1) function was blocked, a process that was greatly accelerated by pre-exposure of this substrate to autologous plasma under flow. These results implicate a collagen receptor(s) on platelets other than alpha(2)beta(1) that can selectively engage domains in banded, but not nonbanded type I collagen when alpha(2)beta(1) function is blocked. In addition, collagen structure may regulate the extent and affinity of the binding under flow of plasma components such as von Willebrand factor and/or other alpha(IIb)beta(3) ligands.     

Age dependence of the biosynthesis of intercellular matrix macromolecules of rabbit aorta in organ culture and cell culture.

The age dependence of the relative rate of biosynthesis of intercellular matrix macromolecules was studied in organ culture and cell culture obtained from aortas of newborn, young and adult rabbits. In organ culture there was a strong decrease with age of the rate of incorporation of (14C)-lysine and (3H)-glucosamine in all macromolecular fractions. Neosynthesis of elastin could be demonstrated by the isolation of labelled demosine at all ages. In cell cultures derived from newborn and adult aortas, no decrease in total incorporation was noticed. The pattern of synthesis and secretion of glycosaminoglycans and glycoproteins did however change with age. These results suggest the existence of matrix-dependent and of a matrix-independent regulation of the relative rate of synthesis of matrix macromolecules.     

Enhanced platelet adhesion to collagen in scleroderma. Effect of scleroderma plasma and scleroderma platelets

The effect of plasma on platelet adhesion to collagen coated microtiter wells was investigated in 22 patients with scleroderma and 26 control subjects. In the control subjects, platelet adhesion was 38 +/- 13% (mean +/- SD) of adhesion with buffer alone; in scleroderma patients adhesion was 64 +/- 20% (p less than 0.001). No correlation was seen between the effect of plasma on platelet adhesion to collagen and the plasma levels of either FVIII/von Willebrand factor antigen or fibronectin in either scleroderma or control subjects. Furthermore, scleroderma platelets demonstrated enhanced adhesion compared to control platelets when tested in the presence of either control or scleroderma plasma.     

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)     

Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture.

Two cell lines--Madin-Darby canine kidney (MDCK) and normal murine mammary gland (NMuMG)--growing as monolayers on collagen gels were overlaid with another collagen gel. The cells responded to the overlay by undergoing reorganization resulting in the creation of lumina. MDCK cells formed lumina that coalesced to form large cavities comparable in size with a tubule. NMuMG cells formed clusters surrounding small lumina, which appeared similar to acini of glandular tissue. The characteristic arrangements, described here by light and electron microscopy, resembled the morphology of the tissues of cell line origin. MDCK cells, grown in the presence of serum, formed lumina whether or not serum was removed at the time of overlay, whereas NMuMG cells required either a nondialyzable component of serum or hormonal supplements in serum-free defined media. Lumen formation was delayed by MDCK cells in the presence of the glutamine analog 6-diazo-5-oxo-L-norleucine, but this compound did not affect NMuMG lumen formation. In both cell lines, lumen formation was unaffected by the absence of sulfate, the presence of an inhibitor of sulfated glycosaminoglycan synthesis, or an inhibitor of collagen synthesis. DNA synthesis accompanied lumen formation but was not required.     

Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen

Platelet responses to collagen are mediated by the combined actions of the integrin alpha2beta1, which serves as a major collagen-binding receptor, and the GPVI/FcRgamma-chain complex, which transmits collagen-specific activation signals into the cell interior through the action of an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain of the FcRgamma-chain. Despite much progress in identifying components of the signaling pathway responsible for collagen-induced platelet activation, virtually nothing is known about the regulatory elements that modulate this important hemostatic event. PECAM-1, a recently recognized member of the inhibitory receptor family, contains a functional immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain that, when tyrosine phosphorylated, recruits and activates the protein-tyrosine phosphatase, SHP-2. To test the hypothesis that PECAM-1 functions to regulate GPVI/FcRgamma-chain-mediated platelet activation, the responses of wild-type versus PECAM-1-deficient murine platelets to GPVI-specific agonists were compared. Four distinct GPVI/FcRgamma-chain-dependent responses were found to be significantly exaggerated in platelets derived from PECAM-1-deficient mice, including Mg++-independent adhesion to immobilized fibrillar collagen, collagen-induced platelet aggregation, platelet aggregation induced by the GPVI-specific agonist collagen-related peptide, and GPVI/FcRgamma-chain-induced dense granule secretion. Together, these data provide compelling evidence that PECAM-1 modulates platelet responses to collagen, and they implicate this novel member of the inhibitory receptor family in the regulation of primary hemostasis.     

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.     

Modulation of leukemic cell sensitivity to lymphokine-activated killer cytolysis: role of intercellular adhesion molecule-1.

The role of CD11/CD18 leukocyte adhesion molecules and their ligands in mediating non-major histocompatibility complex (MHC) restricted lymphocyte cytotoxicity is controversial. In order to examine the role of target cell intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of lymphocyte function-associated antigen (LFA-1) (CD11a/CD18), we exposed the human leukemia cell line, HL-60, to a variety of agents implicated in modulating ICAM-1 expression and/or sensitivity to lymphocyte cytolysis. Exposure of HL-60 cells to retinoic acid (RA), interferon (IFN)-alpha, IFN-beta, and IFN-gamma induced protection from lymphokine-activated killer (LAK) cytolysis. Only RA and IFN-gamma induced ICAM-1 expression. Tumor necrosis factor and vitamin D3, which also induced ICAM-1 expression, increased HL-60 sensitivity to LAK lysis. Granulocyte-macrophage colony-stimulating factor also increased sensitivity to LAK lysis; ICAM-1 was not induced. The state of cellular differentiation and expression of class I and II MHC antigens also did not correlate with sensitivity to LAK cytolysis. Exposure of untreated HL-60 cells and HL-60 cells expressing ICAM-1 to monoclonal antibody (mAb) versus ICAM-1 did not modulate LAK sensitivity. Exposure of LAK cells to mAb versus LFA-1 partially inhibited cytolysis; mAb versus CD18 inhibited cytolysis more completely. HL-60 cells were resistant to natural killer lysis; exposure to the various experimental agents did not alter sensitivity. We conclude that leukemic cell sensitivity to LAK cytolysis can be modulated by a variety of agents. Although our results suggest a role for leukocyte CD11/CD18 adhesion molecules in LAK cytolysis, the poor correlation between ICAM-1 expression and sensitivity to LAK lysis suggest that interactions other than LFA-1/ICAM-1 conjugation may be more central to the processes involved.     

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.The dynamic connection between the forces generated in the actomyosin cytoskeleton and integrin-mediated focal adhesions (FAs) to the extracellular matrix (ECM) is essential for many physiological processes including cell migration, vascular formation and function, the immune response, and tissue morphogenesis. These diverse functions are mediated by distinct cellular structures including protruding lamellipodia containing nascent FAs that mediate haptotaxis (1), ventral adhesive actin waves that mediate leukocyte transmigration through endothelia (2, 3), and stress fibers (SFs) and FAs that drive fibrillarization of ECM in developing embryos (4, 5). The coordination and interdependence of actin and integrin-based adhesion in these specialized cellular structures are rooted in their biochemical interdependence. Activation of integrins to their high-affinity ECM binding state requires the actin cytoskeleton (6). In turn, integrin engagement with ECM induces signaling that mediates actin polymerization and contractility downstream of Rho GTPases (6, 7). ECM-engaged integrins also affect cytoskeletal organization by physically linking the contractile actomyosin system to extracellular anchorage points (7). Thus, adhesion-associated actin structures are integrated systems that mediate cellular functions requiring coordination of intracellular cytoskeletal forces with ECM binding.Mesenchymal cells generally possess two main types of adhesion-associated actin structures: protruding lamellipodia containing nascent FAs at the cell edge and linear actin bundles in the cell body connected to FAs. Compared with architecturally invariant lamellipodia, adhesion-associated actin bundle structures, including filopodia, the perinuclear actin cap/transmembrane actin-associated nuclear lines, trailing edge bundles, and dorsal SFs, are more diverse in their morphology and less well understood in their architecture and function (8–10). The most-studied actin bundle structure is perhaps dorsal SFs, noncontractile bundles associated at one end with a ventral FA near the cell edge and that extend radially toward the cell center and join with dorsal actin arcs on their other end. How the functional specificity of dorsal SFs is generated apart from the many other distinct adhesion-associated actin bundle structures is not well understood.The functional specificity of adhesion-associated actin structures could be generated either on the adhesion side by compositional differences in FA proteins or on the actin side by differences in the nucleation mechanism and actin binding proteins. On the adhesion side, it is well known that different integrin family members bind distinct types of ECM (11, 12). However, cells adhered to different ECMs all form common structures including lamellipodia, filopodia, and multiple types of SFs. In addition to different integrins, FA function could be regulated by the process of “maturation” in which FAs undergo stereotypical dynamic changes in composition and morphology driven by actomyosin-mediated cellular tension (13, 14). Nascent FAs contain integrins, focal adhesion kinase (FAK), a-actinin, and paxillin (13, 15). When tension is applied, nascent FAs grow and recruit hundreds of proteins, including talin, vinculin, and zyxin (16). These mature FAs then either disassemble or further mature into tensin-containing fibrillar FAs that are responsible for fibronectin fibrillogenesis (17). Thus, the changes in FA size and protein content that accompany FA maturation could give rise to functional specialization of adhesion/actin systems.On the other hand, actin filaments in migrating cells are generated by two main classes of nucleators: the Arp2/3 complex and formins (18). Different nucleating proteins generate different actin organization and geometries, which could in turn dictate functional specificity of adhesions. Arp2/3 forms the branched network in lamellipodia and is thought to be linked to nascent FAs through interaction with FAK (19–21) or vinculin (22). The formin family of actin nucleators, which generates linear actin bundles (23), is more diverse, although formins share a common actin assembly core domain (24), (25). Recent work has begun to ascribe the generation of particular actin structures to some of the 15 formins in mammalian cells, particularly members of the diaphanous family and FHOD1 (26–29). Specifically regarding dorsal SFs, evidence points strongly to polymerization by a formin family member (23, 30–32) but no formin has ever been localized to these SFs or their associated FAs in motile cells. Thus, although formins are clearly critical for forming distinct actin structures, whether they cooperate with FA proteins to specify the function of adhesion-associated actin structures in the cell is unclear.We hypothesized that inverted formin 2 (INF2), found in our recent FA proteome (33), may play a critical role in the formation and functional specificity of adhesion-associated actin structures. INF2 is expressed in cells in two isoforms, one containing a membrane-targeting CAAX-motif that plays a role in mitochondrial fission (34) and a non-CAAX isoform whose function is not well characterized. INF2 is an unusual formin insofar as it contains, in addition to the FH1–FH2 domains that polymerize actin, a WH2-like domain at the C terminus (35) that binds actin monomers to regulate autoinhibition, and also mediates filament severing (35, 36). INF2 also interacts with and inhibits members of the diaphanous family of formin proteins (37). INF2 therefore could have multiple possible roles at FAs in local modulation of actin.Here we explore the role of INF2 in mouse embryonic fibroblasts (MEFs). We find for the first time to our knowledge strong localization of an endogenous formin to FAs at the distal tips of dorsal SFs where it is required for actin polymerization at FAs to form dorsal SFs. We show that INF2 plays a role in controlling morphological, but not compositional maturation of FAs. Strikingly, INF2 is responsible for the formation of one specific class of FAs, the fibrillar FAs that organize the ECM; disruption of INF2 leads to defects in ECM fibrillogenesis. Thus, our study demonstrates that INF2 mediates the formation of dorsal SFs and fibrillar FAs, which together comprise a specific integrated adhesion-associated actin structure responsible for the fibrillogenesis of ECM by fibroblasts.     

Nitric oxide inhibits platelet adhesion to collagen through cGMP-dependent and independent mechanisms: The potential role for S-nitrosylation

Nitric oxide (NO)-mediated inhibition of platelet function occurs primarily through elevations in cGMP, although cGMP-independent mechanisms such as S-nitrosylation have been suggested as alternative NO-signaling pathways. In the present study we investigated the potential for S-nitrosylation to act as a NO-mediated cGMP-independent signaling mechanism in platelets. The NO-donor, S-nitrosoglutathione (GSNO), induced a concentration-dependent inhibition of platelet adhesion to immobilized collagen. In the presence of the soluble guanylyl cyclase inhibitor, ODQ, NO-mediated activation of the cGMP/protein kinase G signaling pathway was ablated. However, ODQ failed to completely abolish the inhibitory effect of NO on collagen-mediated adhesion, confirming that cGMP-independent signaling events contribute to the regulation of platelet adhesion by NO. Biotin-switch analysis of platelets demonstrated the presence of several S-nitrosylated proteins under basal conditions. Treatment of platelets with exogenous NO-donors, at concentrations that inhibited platelet adhesion, increased the number of S-nitrosylated bands and led to hyper-nitrosylation of basally S-nitrosylated proteins. The extent of S-nitrosylation in response to exogenous NO was unaffected by platelet activation. Importantly, platelet activation in the absence of exogenous NO failed to increase S-nitrosylation beyond basal levels, indicating that platelet-derived NO was unable to induce this type of protein modification. Our data demonstrate that S-nitrosylation of platelet proteins in response to exogenous NO may act as a potentially important cGMP-independent signaling mechanism for controlling platelet adhesion.     

Significance of platelet endothelial cell adhesion molecule-1 (PECAM-1) and intercellular adhesion molecule-1 (ICAM-1) expressions in preeclamptic placentae

Although preeclampsia (PE) is one of the most important problems affecting pregnant women, etiologic factors in its development are still unclear. We aimed to investigate the expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and intercellular adhesion molecule-1 (ICAM-1) in preeclamptic and control healthy placentas. Placental tissue samples were obtained after delivery from patients diagnosed with PE, and from normal term pregnants and analyzed by immunohistochemistry for the expression levels of the two adhesion molecules PECAM-1 and ICAM-1. A strong expression of PECAM-1 in endothelial cells lining the vessel walls of placental villi in placentas of control group was found, but the intensity of PECAM-1 expression was highly reduced in placentas of PE group (p = 0.017). Conversely, a strong expression of ICAM-1 was observed in placental villi in PE, significantly higher than that of normal placentas (p = 0.005). The findings of a decrease of PECAM-1 expression and an increase of ICAM-1 expression in preeclamptic placenta suggest the existence of functional roles of these adhesion molecules in the pathophysiology of PE, probably by contributing to the reduced trophoblast invasion and the increased vascular damage, respectively. Inhibiting ICAM-1 (i.e., with ICAM-1 monoclonal antibody) and promoting PECAM-1 expression may be good therapeutic approaches to prevent PE symptoms in the future.     

Activated and unactivated platelet adhesion to monocytes and neutrophils.

To examine the possible receptor-ligand pairs mediating adhesion of activated and "unactivated" platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte-platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP-140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet-heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes.     

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.