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.     

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.     

Morphological study of platelet adhesion dynamics under whole blood flow conditions

A flow system consisting of a parallel-plate flow chamber mounted on the epifluorescence video microscope has been constructed to allow direct visualization of the entire platelet adhesion process under whole blood flow conditions. Adhered platelets with recorded adhesion history were individually identified and observed in detail using a scanning electron microscope. In this study we used cover glasses coated with fibrinogen, fibrin, or collagen as the testing surface. From experiments carried out at the surface shear rate of 445 s(-1), we found that (1) platelet adhesion was a dynamic process that involved attaching, detaching, relocation and transient contact; (2) platelets adhered to all three types of protein-coated surfaces with platelet adhesion on collagen being most unstable; (3) most of these adhered platelets immediately formed short pseudopods after surface contact; (4) platelets adhered to fibrinogen or fibrin were basically non-overlapping and they underwent further shape change with increasing number /length of pseudopods and increasing extent of cytoplasmic spreading; (5) on collagen-coated surface most incoming platelets attached to previously adhered platelets rather than to the collagen threads for blood-surface contact times longer than 30 s; (6) these platelets formed multicellular thrombi with largest thrombi located at about 0.2-0.4 mm from the upstream edge and (7) platelets in the thrombi formed numerous short pseudopods and started fusing with one another within 2 min. These observations show that platelet adhesion under blood flow is a complex and dynamic process and that adhered platelets undergo heterogeneous post-contact morphological changes. Moreover, our results indicate that fibrinogen and fibrin coatings are adhesive while collagen coating is most stimulatory to platelets.     

Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions

Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood ( approximately 5 x 10(4) platelets/microL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)-labeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.     

Role of cyclic nucleotides in rapid platelet adhesion to collagen

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.     

Activation of protein kinase C is required for the stable attachment of adherent platelets to collagen but is not needed for the initial rapid adhesion under flow conditions

We have investigated the role of protein kinase C (PKC) in the initial events of alpha(2)beta(1)-integrin-mediated platelet adhesion to collagen under flow conditions. Although adhesion caused activation of PKC, as evidenced by pleckstrin phosphorylation, the PKC inhibitors GF 109203X and G? 6976 had no effect on adhesion, even though they prevented pleckstrin phosphorylation. The initial kinetics and extent of platelet adhesion to collagen (<5 seconds) and tyrosine phosphorylation of p125(FAK) and p72(syk) were not influenced by the PKC inhibitors, whereas adhesion to polylysine was prevented. These results indicate that adhesion to collagen and polylysine involve different mechanisms and requirements for PKC activation. Pretreatment with GF 109203X destabilized collagen-adherent platelets, accelerating their detachment, which was associated with tyrosine dephosphorylation of p125(FAK). Thus, although PKC activation was not required for rapid platelet adhesion to collagen, it appears to play an important role in stabilizing the attachment of adherent platelets to collagen. We also examined the effect of PKC activation by the phorbol ester phorbol 12-myristate 13-acetate (PMA) on platelet adhesion to collagen. PMA at 100 nmol/L strongly potentiated adhesion and tyrosine phosphorylation of p125(FAK) and p72(syk) and activated beta(1)-integrins, as determined by increased exposure of the 15/7 epitope. The PMA-stimulated adhesion was partially blocked by an anti-alpha(2)beta(1) antibody, was completely inhibited by GF 109203X, and was not correlated with the extent of pleckstrin phosphorylation. Therefore, strong PKC activation may lead to inside-out signaling, enhancing the role of beta(1)-integrins in adhesion. Pleckstrin phosphorylation does not appear to be involved in the initial phase of basic or PMA-stimulated adhesion but may help stabilize the adherent platelets.     

Platelet adhesion to collagen in individuals lacking glycoprotein IV

The Naka isoantigen is expressed on glycoprotein (GP) IV (CD36), a platelet membrane GP that has been identified as having a role in platelet interactions with collagen and thrombospondin and in binding Plasmodium falciparum-infected erythrocytes to endothelial cells and melanoma cells. We have studied normal platelets and Naka- platelets from two Japanese donors that have 1% of GPIV by concentration- dependent antibody binding and flow cytometry. We studied the adherence of normal and Naka- platelets to types I, III, and IV collagen in static and to type I collagen in flowing systems at high shear force. We have also studied aggregation of normal and Naka- platelets to type I collagen. Naka- platelets showed normal or increased aggregation to type I collagen and normal adhesion to types I, III, and IV collagen in the presence of Mg++ or EDTA. Platelet aggregation and adhesion were inhibited by the anti-alpha 2 beta 1 antibody 176D7 to the same extent in Naka- as in normal platelets. We also studied endogenous thrombospondin surface expression and found that thrombin-stimulated Naka- platelets expressed the same amount of thrombospondin as did normal platelets. From our studies with Naka- platelets, we cannot identify a definitive role for GPIV in platelet aggregation, in adhesion to types I, III, and IV collagen, or in endogenous thrombospondin binding to platelets.     

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.     

Adhesion of platelets to surface-bound fibrinogen under flow

After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.     

Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (alpha 2 beta 1-integrin)

Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.     

Platelet adhesion to cyanogen-bromide fragments of collagen alpha 1(I) under flow conditions

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.     

Adhesion efficiency, platelet density and size

We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low-density (1.040 < d < 1.065 g/ml), intermediate-density (1.065 < d < 1.070 g/ml) and high-density (1.070 < d < 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous-flow, micro-affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGI2). In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial-flow conditions. The greater efficiency of high-density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.     

Shear-dependent functions of the interaction between soluble von Willebrand factor and platelet glycoprotein Ib in mural thrombus formation on a collagen surface

Recent flow studies have clearly established the function of von Willebrand factor (vWF) in initial platelet adhesion, in which the interaction of surface-immobilized vWF with platelet glycoprotein (GP) Ib, particularly at high shear rates, leads to the transient capture of flowing platelets onto the surface. This interaction is thought to trigger activation of platelet GP IIb/IIIa, leading to irreversible platelet adhesion and subsequent mural thrombus growth. The role of vWF-GP Ib interaction in secondary thrombus growth remains to be clarified, however. In this study, time-course images of the thrombus formation process were obtained using a whole blood flow system that allows real-time visualization of fluorescence-labeled platelet thrombus formation. The system employs a collagen-coated surface in a parallel plate flow chamber mounted on an epifluorescence microscope, which is then subjected to computer-assisted image analysis. In perfusion of blood preincubated with anti-vWF antibody NMC-4, which blocks the vWF-GP Ib interaction in solution, neither primary platelet adhesion nor subsequent thrombus growth on a collagen surface was detected at high shear rates (> or = 1210/s). In addition, even under experimental conditions in which initial platelet adhesion normally occurred, NMC-4-treated blood perfusion showed an apparent defect of secondary thrombus growth at the same high shear rates. The overall process of thrombus formation at low shear rates (< or = 340/s) was not affected by specific blockers of the vWF-GP Ib interaction. These findings indicate that, in addition to the interaction of surface-immobilized vWF with GP Ib in platelet adhesion, the interaction of soluble vWF with GP Ib is required for secondary thrombus growth selectively at high shear rates.     

Platelet adhesion and aggregation on human type VI collagen surfaces under physiological flow conditions

Type VI collagen is a subendothelial constituent that binds von Willebrand factor (vWF) and platelets. The interaction of platelets with type VI collagen and the roles of platelet glycoprotein (GP) receptors and vWF were studied under flow conditions using epi- fluorescent videomicroscopy coupled with digital image processing. We found that surface coverage was less than 6% on collagen VI at a relatively high-wall shear rate (1,000 s-1) and was approximately 60% at a low-wall shear rate (100 s-1). The molecular mechanisms involved in low-shear platelet binding were studied using monoclonal antibodies to platelet GPIb and GPIIb-IIIa, and polymeric aurin tricarboxylic acid. Anti-GPIIb-IIIa was the most effective in eliminating adhesion (surface coverage, 0.8%), followed by anti-GPIb (4.3%), and ATA (12.6%). Experiments with von Willebrand disease blood indicate that vWF is involved in platelet adhesion to collagen VI at 100 s-1. In the absence of vWF, there may be direct binding of platelet GPIIb-IIIa complexes to collagen VI. Adhesion and aggregation on collagen VI are different in shear rate dependence from collagen I. Our results suggest a possible role for collagen VI and vWF in platelet adhesion and aggregation in vascular regions with low shear rates.     

Inhibition of platelet adhesion to collagen by monoclonal anti-CD36 antibodies

Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non-denaturing procedure. These antibodies reacted with control platelets, but not Nak^a-negative platelets which lack CD36, as measured by flow cytometry and by immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg²⁺-independent conditions but had little effect in the presence of Mg²⁺. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800 s^?1), whereas 131.5 was without effect. These are the first anti-CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet–collagen interaction.     

Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.     

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF- GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.     

Platelet receptors for collagens

Collagens belong to the constituents that determine the thrombogenicity of the vessel wall. Seven genetically distinct collagens-type I, III, IV, V, VI, VIII and XIII-have been identified in the vessel wall. The interaction of platelets with collagens is a complex process since collagens are not only potent platelet agonists but also adhesive proteins. In recent years several platelet membrane glycoproteins have been shown to be involved in platelet-collagen interactions. The mechanisms of platelet-collagen interaction can be divided into primary, direct interactions and secondary, indirect interactions. A number of direct receptors for the collagens have been proposed. The glycoprotein complex Ia/IIa, also called VLA(2), α(2)β(1) integrin or CD49b/CD29, meets several criteria as the major platelet receptor for different types of collagens. There is some evidence that glycoprotein IIIb, also called glycoprotein IV or CD36, functions at the earliest stages of collagen adhesion as a platelet collagen receptor. CD36 seems to be essential for type V collagen-induced platelet aggregation and adhesion. Another putative platelet-collagen receptor is P62, a glycoprotein with a molecular weight of 62 kDa under reducing conditions.     

Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.     

A single high-affinity binding site for von Willebrand factor in collagen III, identified using synthetic triple-helical peptides

The essential event in platelet adhesion to the injured blood vessel wall is the binding to subendothelial collagen of plasma von Willebrand factor (VWF), a protein that interacts transiently with platelet glycoprotein Ibalpha (GPIbalpha), slowing circulating platelets to facilitate firm adhesion through collagen receptors, including integrin alpha2beta1 and GpVI. To locate the site in collagen that binds VWF, we synthesized 57 overlapping triple-helical peptides comprising the whole triple-helical domain of collagen III. Peptide no. 23 alone bound VWF, with similar affinity to that of native collagen III. Immobilized peptide no. 23 supported platelet adhesion under static and flow conditions, processes blocked by an antibody that prevents collagen from binding the VWF A3 domain. Truncated and alanine-substituted peptides derived from no. 23 either strongly interacted with both VWF and platelets or lacked both VWF and platelet binding. Thus, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF-binding sequence in collagen III.