Magnesium maintains endothelial integrity, up-regulates proteolysis of ultra-large von Willebrand factor, and reduces platelet aggregation under flow conditions

2008 ◽  
Vol 99 (03) ◽  
pp. 586-593 ◽  
Author(s):  
Miguel Cruz ◽  
Khatira Aboulfatova ◽  
Cecilia Martin ◽  
Hiuwan Choi ◽  
Angela Bergeron ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Umbilical Cord ◽  
Flow Conditions ◽  
Endothelial Functions ◽  
Endothelial Integrity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Insight Into

SummaryMg ++ regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood.We investigated the roles of MgSO4 in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation.Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm2. Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO4. Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO4 on platelet adhesion and aggregation. Also studied were the effects of MgSO4 on ristocetin-induced platelet aggregation andVWF-collagen interaction.Maintenance of endothelial integrity required physiological levels of MgSO4, but exogenous MgSO4 showed no additional benefits.Exogenous MgSO4 significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions.This effect is likely to be mediated through VWF as Mg++ partially inhibited ristocetin-induced platelet aggregation andVWF binding to collagen.MgSO4 is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy.

scholarly journals Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1336-1340 ◽  
Author(s):  
G Escolar ◽  
A Cases ◽  
E Bastida ◽  
M Garrido ◽  
J Lopez ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Indirect Evidence ◽  
Flow Conditions ◽  
Experimental Conditions ◽  
Shear Rates ◽  
Functional Defect ◽  
Uremic Patients ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de- endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

scholarly journals Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 546-550 ◽  
Author(s):  
J McPherson ◽  
S Brownlea ◽  
MB Zucker
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Retention Test ◽  
Platelet Adhesion ◽  
Glass Beads ◽  
Two Stage ◽  
Second Stage ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one- stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP- induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1149-1155 ◽  
Author(s):  
Mitsuhiro Kuwahara ◽  
Mitsuhiko Sugimoto ◽  
Shizuko Tsuji ◽  
Shigeki Miyata ◽  
Akira Yoshioka
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Cytosolic Calcium ◽  
Flow Chamber ◽  
Flow Conditions ◽  
Color Changes ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Calcium Green

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Unique Antiplatelet Effects of a Novel S-Nitrosoderivative of a Recombinant Fragment of von Willebrand Factor, AR545C: In Vitro and Ex Vivo Inhibition of Platelet Function

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1693-1700
Author(s):  
Aida Inbal ◽  
Osnat Gurevitz ◽  
Ilia Tamarin ◽  
Regina Eskaraev ◽  
Angela Chetrit ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Aggregate Formation ◽  
Recombinant Fragment ◽  
Von Willebrand ◽  
Willebrand Factor

The recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC50 = 0.02 ± 0.006 μmol/L v 0.1 ± 0.03 μmol/L, P = .001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate–induced platelet aggregation (IC50 = 0.018 ± 0.002 μmol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 μmol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P ≤ .004 for each parameter by ANOVA). In an ex vivo rabbit model, we also found that S-nitroso-AR545C had a more marked and more durable inhibitory effect on botrocetin-induced platelet aggregation than did AR545C, and these differences were also reflected in the extent and duration of effect on the prolongation of the bleeding time in these animals. These data show that S-nitroso-AR545C has significant and unique antiplatelet effects, inhibiting both adhesion and aggregation, by blocking platelet GPIb receptor through the AR545C moiety and elevating platelet cyclic 3′,5′-guanosine monophosphate through the -SNO moiety. These observations suggest that this NO-modified fragment of vWF may have potential therapeutic benefits as a unique antithrombotic agent.

scholarly journals Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 345-353 ◽  
Author(s):  
RR Hantgan ◽  
G Hindriks ◽  
RG Taylor ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Rates ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

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

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1035-1042 ◽  
Author(s):  
H Lankhof ◽  
YP Wu ◽  
T Vink ◽  
ME Schiphorst ◽  
HG Zerwes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Static Condition ◽  
Site Directed Mutagenesis ◽  
Flow Conditions ◽  
Rgd Sequence ◽  
Absolute Requirement ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
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.

Biomechanical Properties of Bonds Mediated by the von Willebrand Factor A1 Domain during Platelet Adhesion under Flow Conditions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3665-3665
Author(s):  
Marianna M. Machin ◽  
Jennifer N. Orje ◽  
Paolo Canu ◽  
Zaverio M. Ruggeri
Keyword(s):  
Residence Time ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Bond Formation ◽  
Biomechanical Properties ◽  
Hydrodynamic Forces ◽  
Flow Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The initial attachment of platelets to vascular lesions exposed to a high wall shear rate (γw) depends on the interaction between the membrane glycoprotein (GP) Ibα, a component of the GP Ib-IX-V receptor complex, and the A1 domain of surface-immobilized von Willebrand factor (VWFA1). We performed perfusion experiments under different flow conditions to measure transient platelet contacts onto immobilized recombinant VWFA1 and determine the probability of bond formation (capturing) and resistance to tensile stress (bond lifetime) of VWFA1-GP Ibα interactions. To define how molecular conformations influence the biomechanical properties of the bonds, we compared fragments exhibiting the native dimeric assembly of A1 domain with monomeric fragments obtained by selective purification of recombinant protein expressed in stable D. melanogaster cell lines. The minimum coating concentration of dimeric VWFA1 at which platelet adhesion events were statistically significantly different from nonspecific interactions on uncoated glass was 1 μg/ml. In the range of γw between 30 and 30,000 s−1, there was no threshold value for the initiation of adhesion, as seen for selectins. The number of adhering platelets first increased and then decreased with monotonically increasing γw, indicating the effect of transport phenomena as well as hydrodynamic forces on the VWFA1-GP Ibα interaction. Maximum event number was at 5,000 s−1 for dimeric and 1,500 s−1 for monomeric VWFA1. The platelet count had no statistically relevant influence on the efficiency of capturing and duration of adhesive contacts. As γw increased, a higher coating concentration of the VWF A1 domain was required to initiate platelet adhesion. The coating concentration determined the number of individual adhesion events that could occur over a defined period of time but did not affect the residence time, which is a measure of the strength of the bond between the receptor and the ligand. Hydrodynamic forces generated by blood flow shortened the duration of VWF-GP Ibα interactions. The percentage of platelets that had a residence time of less than 0.1 s increased almost linearly with increasing γw. Dimeric A1 domain was more efficient than the monomeric counterpart in promoting platelet adhesion as it displayed activity at lower coating concentrations. At permissible γw, a 10-fold higher monomer than dimer coating concentration (2 vs. 20 μg/ml) was required to obtain a similar capturing efficiency. Moreover, the upper limit of γw compatible with the initiation of adhesion was significantly higher for dimeric as compared to monomeric A1 domain. Doubling the dimer coating concentration resulted in a 5-fold increase in the γw limit for adhesion, but the same increase in the monomer coating concentration did not enhance the probability of bond formation at higher γw. In spite of the substantial difference in capturing efficiency, monomeric and dimeric VWFA1 supported platelet adhesion events of similar duration at any given γw, indicating that a different molecular conformation did not affect the lifetime of the interaction with GP Ibα. These results indicate that the dimeric assembly of A1 domains in VWF multimers may be crucial to support the initiation of platelet adhesion at high shear rates, but the duration of each adhesion event is limited by intrinsic properties of the individual VWFA1-GP Ibα bond.

Distinct Functional Conformations of von Willebrand Factor A1 Domain in Support of Platelet-Surface or Platelet-Platelet Interactions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5182-5182
Author(s):  
Gianmarco Podda ◽  
James R. Roberts ◽  
Richard A. McClintock ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Platelet Adhesion ◽  
Shear Rates ◽  
Amino Terminal ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Positively Charged

Abstract The adhesive protein, von Willebrand factor (VWF), is generally considered a key substrate for platelet adhesion to the vessel wall, yet its role in platelet cohesion (aggregation) may be equally important for normal thrombus formation. In either case, the function of VWF is mediated by the primary interaction of the VWF A1 domain (VWF-A1) with glycoprotein (GP) Ibα, a component of the GPIb-IX-V receptor complex on the platelet membrane. Because normal plasma VWF in solution and GPIb coexist in circulating blood without any appreciable interaction, it has been postulated that conformational changes occur when VWF becomes immobilized and/or under the effect of pathologically elevated shear stress, such that binding to the receptor becomes possible and resultis in platelet tethering to a surface and shear-induced aggregation. Changes of the molecular shape of VWF, from coiled to extended, have been shown under the effect of hemodynamic forces, but evidence for conformational changes within VWF-A1 has remained elusive. The crystal structure of VWF-A1 in complex with a GPIbα amino terminal fragment has revealed that the VWF-A1 residues involved in the interaction are comprised between positions 544–614 and, in particular, do not include several positively charged Arg and Lys residues located in helices α4 and 5 (residues 627–668). The latter appear as likely candidates to interact with negatively charged residues in GPIbα as a consequence of potential conformational changes induced by tensile stress on the bond following an initial ligand-receptor contact. We tested this hypothesis by evaluating the ability of selected VWF-A1 mutants to support platelet adhesion or aggregation, respectively, under controlled flow conditions. Methods. We expressed in insect cells and purified a series of VWF-A1 fragments comprising residues 445–733. One fragment had native sequence and 8 had single or multiple substitutions of positively charged amino acid residues in helices α4 and/or α5. None of the substituted residues contribute to contacts with GP Ibα in the known crystal structures of the corresponding complex, and all except one were between 8 and 20 angstroms away from the closest GPIbα residue. All the fragments were dimeric (d) owing to the presence of interchain disulfide bond(s). Results: Native dVWF-A1 in solution supported platelet aggregation in a laminar flow field. Of the 8 mutants, 5 had variably decreased function (up to 95% less aggregation) and 2 had increased function (up to 200% increase in aggregation). The same results were observed with platelet-rich plasma in suspension or by measuring platelet aggregate formation with blood cells perfused over immobilized VWF-A1 at wall shear rates as high as 10,000 1/s. In contrast, as judged by the number of tethered platelets and their rolling velocities, all mutants supported adhesion as well as or better that the native VWFA-1 at all shear rates tested (500–25,000 1/s). Conclusions: These results provide structural evidence for the existence of different VWF-A1 conformers that can modulate adhesive properties with distinct effects on platelet adhesion to a surface or platelet aggregation.

Adhesive Domains in the Collagen III Fragment α1(III)CB4 that Support α2β1- and von Willebrand Factor-mediated Platelet Adhesion under Flow Conditions

1999 ◽  
Vol 82 (09) ◽  
pp. 1137-1144 ◽  
Author(s):  
Martin IJsseldijk ◽  
Glenda Heijnen-Snyder ◽  
Eric Huizinga ◽  
Laurence Morton ◽  
C. Graham Knight ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Recognition Site ◽  
Flow Conditions ◽  
Binding Studies ◽  
Collagen Iii ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySeven overlapping peptides derived from the bovine α1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin α2β1-recognition site has been assigned within this fragment to residues 522-528 of the collagen α1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both α2β1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solidphase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*-GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the α1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the α2 subunit of integrin α2β1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the α2β1-recognition site in this locality in α1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the α1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of α2β1-, vWF- and rLAPP-binding sites all in close proximity in α1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

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