scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535 ◽  
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

Cleavage of human von Willebrand factor by platelet calcium-activated protease

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 352-356 ◽  
Author(s):  
TJ Kunicki ◽  
RR Montgomery ◽  
J Schullek
Keyword(s):  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Apparent Molecular Weight ◽  
Aminocaproic Acid ◽  
Crossed Immunoelectrophoresis ◽  
Nonionic Detergent ◽  
Epsilon Aminocaproic Acid ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.

scholarly journals Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1804-1809 ◽  
Author(s):  
JL Miller ◽  
ZM Ruggeri ◽  
VA Lyle
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Normal Platelet ◽  
High Affinity Binding Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed

Abstract The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

LOCALIZATION OF THE PLATELET-BINDING AND HEPARIN-BINDING DOMAINS OF BOVINE VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Edward P Kirby ◽  
Mary Ann Mascelli ◽  
Carol Silverman ◽  
Daniel W Karl
Keyword(s):  
Von Willebrand Factor ◽  
Human Platelets ◽  
Binding Activity ◽  
Heparin Binding ◽  
Amino Groups ◽  
Binding Domains ◽  
Platelet Binding ◽  
Diamondback Rattlesnake ◽  
Von Willebrand ◽  
Willebrand Factor

Bovine von Willebrand Factor (vWF) binds directly to human platelets and also to heparin-agarose. Cleavage of vWF with Protease I, a metalloenzyme isolated from the venom of the western diamondback rattlesnake, produces two major fragments with apparent Mr of 250 kD and 200 kD. The 200 kD fragment competes with native vWF for binding to the GPIb-associated vWF receptor on formalin-fixed human platelets and has weak platelet-agglutinating activity. It is composed of three polypeptide chains of apparent Mr of 97 kD, 61 kD, and 35 kD. Monoclonal antibodies #2 and H-9, which inhibit binding of vWF to a GPIb-associated receptor of platelets, recognize the 200 kD fragment.Modification of vWF with ^5x-la.beled. Bolton-Hunter reagent (I*-BHR) causes inhibition of platelet-agglutinating activity at very low levels of incorporation. Modification of less than 2% of the amino groups in vWF causes 50% loss of platelet agglutinating activity and a decreased affinity of vWF for binding to platelets. Labeling with I*-BHR does not block binding to heparin-agarose, even when 5-10% of the amino groups are modified. Differential labeling at pH 7.0 and pH 8.5, followed by proteolytic fragmentation with Protease I, suggests that it is the modification of amino groups on the 200 kD fragment which is responsible fpr the decrease in platelet binding activity. Modification of the 97 kD peptide chain is best correlated with this loss of platelet binding activity.Heparin inhibits the agglutination of human platelets by bovine vWF. The 200 kD fragment of vWF binds both to platelets and to heparin-agarose. These observations suggest that the heparin-binding and platelet-binding domains of vWF, although distinct from one another, reside in the same region of the vWF molecule. The platelet-binding domain contains a small number of very reactive amino groups which are required for vWF binding to human platelets.These studies were supported by a grant from the National Institutes of Health (#HL27993).

scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366 ◽  
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

scholarly journals Proteolysis of platelet glycoprotein Ib by plasmin is facilitated by plasmin lysine-binding regions

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1280-1284 ◽  
Author(s):  
B Adelman ◽  
AD Michelson ◽  
J Greenberg ◽  
RI Handin
Keyword(s):  
Binding Sites ◽  
Direct Interaction ◽  
Aminocaproic Acid ◽  
Platelet Glycoprotein ◽  
Binding Region ◽  
Glycoprotein Ib ◽  
Platelet Membrane Receptor ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

We have characterized the effects of plasmin on glycoprotein Ib (GpIb), a platelet membrane receptor for von Willebrand factor (vWF), and on glycocalicin, a fragment of the alpha chain of GpIb that contains the vWF-binding region. The addition of 4.5 X 10(-7) mol/L plasmin to washed platelets caused a time-dependent decrease in ristocetin- induced, vWF-dependent platelet agglutination. epsilon-Aminocaproic acid (EACA) inhibited plasmin release of glycocalicin-related antigen from washed platelets and preserved vWF-dependent platelet agglutination, thus indicating that the lysine-binding sites on plasmin facilitated its degradation of GpIb. To demonstrate a direct interaction between plasmin and the vWF-binding region of GpIb we incubated purified glycocalicin with plasmin. Plasmin degraded the glycocalicin into two small carbohydrate-poor peptides and into a larger carbohydrate-rich fragment. EACA was able to inhibit plasmin- mediated degra dation of glycocalicin in a concentration-dependent fashion. These studies indicated that plasmin degradation of GpIb was due to a direct interaction between plasmin and GpIb and that this effect was mediated by the lysine-binding region of the plasmin molecule.

Identification of the von Willebrand Factor Binding Site in Collagen Using Triple Helical Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 413-413
Author(s):  
Ton Lisman ◽  
Nicholas Raynal ◽  
Dafna Groeneveld ◽  
Ben Maddox ◽  
Tony Peachey ◽  
...  
Keyword(s):  
Binding Site ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Collagen Type Iii ◽  
Collagen Binding ◽  
Type Iii ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Molecular Description

Abstract The interaction of the plasma protein von Willebrand factor (VWF) with subendothelial collagen initiates adhesion of blood platelets to the damaged vessel wall or ruptured atherosclerotic plaque. A detailed molecular description of the VWF-collagen interaction may facilitate development of a novel class of antithrombotic drugs that inhibits this vital step in platelet thrombus formation. We have previously used site-directed mutagenesis to map the collagen-binding site in the VWF A3 domain. Here, we report the identification of a 9-aminoacid sequence in collagen type III which mediates VWF binding. We have synthesized a set of 57 peptides, each containing 27 amino acids of native collagen sequence flanked at each end by five GPP (standard amino acid nomenclature) triplets which support the triple helical structure that is essential for ligand recognition by collagen. The sequence of each peptide overlaps by 9 amino acids with that of each adjacent peptide. A single peptide from this set (#23) was shown to bind VWF in a solid-phase binding assay. The affinity of peptide #23 for VWF was comparable to that of native collagen type III. The peptide #23-VWF interaction was abolished by a monoclonal antibody directed against the collagen-binding site on the VWF A3 domain. Furthermore, recombinant VWF variants that were previously shown to lack collagen-binding capacity (delta A3, His1023Ala) were not able to bind to the peptide. Using surface plasmon resonance, we showed that the peptide bound strongly to the isolated VWF A3 domain (Kd 300 nM). Immobilized peptide #23 also supported platelet adhesion from whole blood under flow conditions and washed platelet adhesion under static conditions. Platelet adhesion to peptide #23 could be abrogated by a monoclonal antibody directed against the VWF A3 domain, which inhibits the interaction of VWF with full-length collagen. We subsequently synthesized a set of truncated and alanine-modified triple helical peptides based on the sequence of #23, which were all tested for VWF and platelet binding from whole blood under flow conditions. Modified peptides either strongly interacted with both VWF and platelets, or lacked both VWF and platelet binding. Based on these experiments, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF binding sequence in collagen type III. Mutation of either Q or M to alanine (A) did not affect VWF binding, whereas replacement of R, O, V, and F by A completely abolished VWF binding. Glycine residues were not replaced, as they are essential for triple helix formation. A model of the VWF A3 domain with this nonapeptide collagen sequence was constructed to give detailed insight into the VWF-collagen interaction. In conclusion, we have identified a 9 amino acid sequence in collagen type III that is entirely responsible for high affinity binding to VWF. The detailed molecular description of the VWF-collagen interaction described here may facilitate development of agents disrupting this interaction, which may have potential as antithrombotic drugs.

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