Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the shortlived vasodilator bradykinin and leaves behind twochain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the ␣ IIb  3 integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486 -Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-␣ IIb  3 -integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486 -Lys-502 abrogated the ␣ IIb  3 -integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486 -Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new antithrombotic compounds may become available on the basis of peptide Gly-486 -Lys-502 of HK domain 5.After vascular injury, the adhesion of platelets to the exposed subendothelial extracellular matrix together with platelet aggregation are essential steps in primary hemostasis. Subsequent fibrin formation not only stabilizes the temporary thrombus but also seals the platelet plug against diffusion of platelet secretory products, such as clotting factors, cell stimulatory agonists, or regulatory growth factors (1). At high shear rates platelets predominantly stick to von Willebrand factor and collagen, whereas under low shear rate or static conditions several other extracellular matrix proteins, such as fibrin and vitronectin (VN) 1 mediate ␣ IIb  3 integrin-dependent platelet adhesion via their Arg-Gly-Asp (RGD) sequence. Furthermore, binding of different agonists, such as ADP, thrombin, or collagen induces signaling events ultimately activating the receptor function of ␣ IIb  3 integrin for soluble fibrinogen leading to platelet aggregation (2-6).Because of its distribution between the flowing blood and the underlying tissues, VN is believed to exhibit several regulatory functions, particularly during vascular remodeling (7,8). The multimeric form of VN becomes incorporated into the vascular extracellular matrix (9, 10), accumula...
SummaryThe alteration of rheological blood properties as well as deterioration of vascular perfusion conditions and cell-cell interactions are major determinants of thrombus formation. Herein, we present an experimental model which allows for quantitative in vivo microscopic analysis of these determinants during both thrombus formation and vascular recanalisation. The model does not require surgical preparation procedures, and enables for repeated analysis of identical microvessels over time periods of days or months, respectively. After i.v. administration of FITC-dextran thrombus formation was induced photochemically by light exposure to individual arterioles and venules of the ear of ten anaesthetised hairless mice. In venules, epiillumination induced rapid thrombus formation with first platelet deposition after 0.59 ± 0.04 min and complete vessel occlusion within 7.48 ±1.31 min. After a 24-h time period, 75% of the thrombosed venules were found recanalised. Marked leukocyte-endothelial cell interaction in those venules indicated persistent endothelial cell activation and/or injury, even after an observation period of 7 days. In arterioles, epi-illumination provoked vasomotion, while thrombus formation was significantly (p <0.05) delayed with first platelet deposition after 2.32 ± 0.22 min and complete vessel occlusion within 20.07 ±3.84 min. Strikingly, only one of the investigated arterioles was found recanalised after 24 h, which, however, did not show leukocyte-endothelial cell interaction. Heparin (300 U/kg, i.v.) effectively counteracted the process of thrombus formation in this model, including both first platelet deposition and vessel occlusion. We conclude that the model of the ear of the hairless mouse allows for distinct in vivo analysis of arteriolar and venular thrombus formation/ recanalisation, and, thus, represents an interesting tool for the study of novel antithrombotic and thrombolytic strategies, respectively.
E rnst F. Lüscher had posed somei nteresting questions in 2000 about the timeflowofthe first platelet reactionsafter activation: shape change (SC) or aggregation (1). Regarding in-vivo studies-c ommencing on the observations of Bizzozeroi n1 882 (2) -L üscherd educed fromt he literaturea nd from owninvestigations (3, 4) thatshape change whichprecedes aggregation would be an in-vitro artefact. Indeed,aplethoraof ultra-structural studies dealt with the examination of aggregated platelets starting with the investigations of Bornetal. 1980(5), and Borna nd manyo thers used transmission electronm icroscopy (TEM)tosupportthe viewthat the initial decrease in light transmission in plateleta ggregometryw as caused by platelet shape change.Ye t, recent morphological findings could attribute aggregatesofdiscoid platelets (designateddiscocytes) to the initialc hange in the aggregometer curve (6). More recently,a shear-dependenti n-vitro aggregation of discoid platelets was demonstrated using differential interferencec ontrast microscopyorscanning electronmicroscopy(7-9). These initiallyunstable aggregatesarise under the formation of membrane tethers between plateletsand an adhesive surface. Tetherformation involves the adhesivef unction of glycoprotein Ib/V/IX complex on vonWillebrand factor.The conversion of aggregatesfrom discoidplatelets into stableaggregatesconsisting of platelets showing shape change requiresthe release of ADP.Apartfrom astudy investigating aggregation in agenetic defective mouse(10), until nownoin-vitro aggregate formation between discoid mammalianp latelets could be observedi nt he TEM. Inspiredb yt he aforementioned implications (1) we investigated whether it is possible to demonstrateaggregation of discoid platelets in vitro . To induce aggregation we chose ADP, an agonist of platelets released fromdamaged cells, particularlyerythrocytes, at sites of vasculari njury( 11). Lüscheri nitiatedt hese investigations and inspiringlya ccompaniedt hem up to his sudden death in April 2002.Results presentedinthis reportindicate that already 1.5 seconds (s) after ADP-stimulation of discoid platelets in-vitro fibrinogen moleculesi nitiatedt he first contactsb etween platelets still retaining their discoid shape.The contacts were seen at any site of the plateletsurface. One second later,shape change took place, and focal contacts were formedo nt he plateletb ody associated with the constricting contractileg el.The existenceo f bridging fibrinogen moleculesduring the aggregation wasconfirmed by immunolabelling with anti-fibrinogen antibody.Thus, plateletaggregation appears to start withdiscoid platelet-fibrinogen contacts under the chosenin-vitro conditions, and the twostage process leads to formation of stable aggregatesofplatelets with achangedshape. Materials andmethodsAntibody Rabbit-antih uman-fibrinogen IgG Dakopatts (Denmark) was used. Platelet preparationPlatelet-rich plasma( PRP) waso btained by centrifugation (16 minutes[ min],2 30 g) from ACDN IH (formula A) anticoagulated venous blood of healthyv olun...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.