scholarly journals Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Haematologica ◽  
2020 ◽  
pp. 0-0
Author(s):  
Thomas Hoefer ◽  
Akshita Rana ◽  
Be’eri Niego ◽  
Shweta Jagdale ◽  
Hugo J. Albers ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Thrombus Formation ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Constant Shear ◽  
Shear Gradient ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Constant Shear Rate

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.

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 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

Abstract 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.

Platelet Release Reaction in vivo in Patients with Ischemic Heart Disease (IHD) After Exercise and its Prevention with Dipyridamole

1979 ◽  
Author(s):  
H. Yamazaki ◽  
T. Motomiya ◽  
T. Sano
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Statistical Significance ◽  
Isometric Exercise ◽  
Voluntary Contraction ◽  
Anti Platelet Drug ◽  
Healthy Control ◽  
Von Willebrand ◽  
Willebrand Factor

Although an interaction between platelets and arteriosclerotic vessel wall is thought to be important in thrombus formation, a little information was obtained in clinical subjects. We have reported that platelet aggregation Increased in patients with IHD after exercise. To analyse the mechanism of this phenomenon, changes in platelet sensitivity to ADP aggregation, plasma von Willebrand factor and beta-thromboglobulin level were measured in 30 IHD and 30 healthy controls before and Immediately after an isometric exercise (handgrip of 50% voluntary contraction for 2 min). Platelet sensitivity and vWF were determined by original methods detecting microscopically the highest dilution of serially two-fold diluted ADP or test plasma mixed with ristocetin to give platelet aggregation. Beta-TG was measured by RIA Kit. An effect of anti-platelet drug was also observed in IHD. The patients with IHD were administered with placebo or dipyridamole (400 mg/day for 4 weeks) in a crossover single blind fashion. Under placebo, platelet sensitivity to aggregation, vWF and beta-TG increased immediately after the exercise with a statistical significance in IHD. In the healthy control and IHD under dipyridamole, these increases were not observed. The phenomenon may suggest that platelets circulating in sclerotic vessels tend to release and are enhanced in reactivity with smaller stimuli than those in healthy. Such changes might be prevented with dipyridamole.

Elevated Threshold Shear Rate for the Dependence of Glycoprotein Ibα-Mediated Platelet Thrombus Formation onto Immobilized von Willebrand Factor in Mouse Blood.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3662-3662
Author(s):  
Patrizia Marchese ◽  
Taisuke Kanaji ◽  
Denisa D. Wagner ◽  
Jerry Ware ◽  
Zaverio M. Ruggeri
Keyword(s):  
Shear Rate ◽  
Collagen Type ◽  
Thrombus Formation ◽  
Collagen Type I ◽  
Type I ◽  
Shear Rates ◽  
Normal Platelet ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The interaction between platelet glycoprotein (GP) Ibα and von Willebrand Factor (VWF) is essential to initiate platelet deposition at sites of vascular injury and sustain platelet thrombus formation when the shear rate exceeds a threshold value. With human blood, the dependence of normal platelet adhesion and aggregation on VWF-GP Ibα function becomes evident at shear rates above 1,000 s−1. In the last several years, mouse models have been increasingly used to study the mechanisms of thrombus formation in circulating blood, and mice deficient in both VWF and GP Ibα have been generated. These animals offer the opportunity to evaluate whether the pathways of platelet adhesion and aggregation mediated by VWF and GP Ibα are equally important in mouse and human blood as well as to define the threshold shear rate at which the function of these pathways may become essential in the mouse circulation. To address this issue, we used an ex vivo perfusion system using fibrillar collagen type I as the thrombogenic surface and a flow chamber in which the shear rate varied according to a predictable function from the inlet to the outlet in relation to the x,y position in the flow path. Thus, wall shear rates between 5,000 at the inlet and 0 s−1 at the outlet could be evaluated in a single experiment, allowing a precise definition of the threshold at which platelet deposition on the surface could initiate. In these studies we used wild type control animals (WT), mice deficient in VWF (VWF-KO) and mice in which most of the extracellular domain of GP Ibα was replaced by a domain of the human interleukin 4 receptor (GPIb-KO/IL-4R). In the latter case, the ligand binding function of GP Ibα was obliterated, but unlike in GP Ib-KO mice platelet morphology and count were essentially normal. Blood was obtained from the retroorbital vein plexus and contained 100 u/ml heparin as an anticoagulant. Experiments were recorded in real time for the visualization of platelet-surface contacts and confocal videomicroscopy was used for the direct measurement of platelet thrombus volume. With normal mouse blood, platelet formed large thrombi throughout the tested range of shear rates. In contrast, with VWF-KO and GPIb-KO/IL-4R blood, thrombus volume was less than 5% of normal at 5,000 s−1, approximately 50% of normal at 3,000 s−1, but entirely normal at 1,500 s−1. Essentially the same results were observed when the extracellular matrix of mouse fibroblasts, which may better represent the complex thrombogenic properties of the vascular wall, was used as a reactive substrate instead of isolated collagen type I. The different threshold shear rate at which VWF and GP Ibα function are essential for thrombus formation with human and mouse platelets may be explained by the smaller size of the latter, which consequently are subjected to a lower drag at equivalent shear rate levels. Moreover, the similar behavior of VWF-KO and GPIb-KO/IL-4R platelets suggests that, under the conditions of these studies, VWF binding is the predominant GP Ibα function required for normal platelet thrombus formation at high shear rates. The present results should allow a more critical evaluation of the findings derived from mouse models of hemostasis and thrombosis.

Von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1741-1746 ◽  
Author(s):  
Jeffrey F. W. Keuren ◽  
Dominique Baruch ◽  
Paulette Legendre ◽  
Cécile V. Denis ◽  
Peter J. Lenting ◽  
...  
Keyword(s):  
Shear Rate ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
High Shear Rate ◽  
High Shear ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractFibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domains that bind to fibrin and to determine their relevance in platelet adhesion. VWF binds specifically to fibrin with an apparent Kd of 2.2 μg/mL. Competition in the presence of 2 complementary fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), indicated that the high affinity binding site for fibrin is located in the C-terminal part, thus distinct from the A domains. Comparison of 2 deleted rVWF (ΔD4B-rVWF, ΔC1C2-rVWF) suggested that the C1C2 domains contained a fibrin binding site. This site is distinct from RGD, as shown by binding of D1746G-rVWF to fibrin. Perfusion studies at high shear rate demonstrated that C1C2 domains were required for optimal platelet adhesion to fibrin. With the use of a VWF-deficient mouse model, it was found that plasma VWF is critical for platelet tethering and adhesion to fibrin. These results suggest a dual role of fibrin-bound VWF in thrombus formation: first, fibrin-bound VWF is critical in the recruitment of platelets by way of glycoprotein (GP) Ib, and, second, it contributes to stationary platelet adhesion by way of binding to activated αIIbβ3.

scholarly journals The distal carboxyl-terminal domains of ADAMTS13 are required for regulation of in vivo thrombus formation

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5323-5329 ◽  
Author(s):  
Fumiaki Banno ◽  
Anil K. Chauhan ◽  
Koichi Kokame ◽  
Jin Yang ◽  
Shigeki Miyata ◽  
...  
Keyword(s):  
Shear Rate ◽  
Genetic Background ◽  
Thrombus Formation ◽  
Short Form ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Terminal Domains

Abstract ADAMTS13 is a multidomain protease that limits platelet thrombogenesis through the cleavage of von Willebrand factor (VWF). We previously identified 2 types of mouse Adamts13 gene: the 129/Sv-strain Adamts13 gene encodes the long-form ADAMTS13 having the same domains as human ADAMTS13, whereas the C57BL/6-strain Adamts13 gene encodes the short-form ADAMTS13 lacking the distal C-terminal domains. To assess the physiologic significance of the distal C-terminal domains of ADAMTS13, we generated and analyzed 129/Sv-genetic background congenic mice (Adamts13S/S) that carry the short-form ADAMTS13. Similar to wild-type 129/Sv mice (Adamts13L/L), Adamts13S/S did not have ultralarge VWF multimers in plasma, in contrast to 129/Sv-genetic background ADAMTS13-deficient mice (Adamts13−/−). However, in vitro thrombogenesis under flow at a shear rate of 5000 s−1 was accelerated in Adamts13S/S compared with Adamts13L/L. Both in vivo thrombus formation in ferric chloride–injured arterioles and thrombocytopenia induced by collagen plus epinephrine challenge were more dramatic in Adamts13S/S than in Adamts13L/L but less than in Adamts13−/−. These results suggested that the C-terminally truncated ADAMTS13 exhibited decreased activity in the cleavage of VWF under high shear rate. Role of the C-terminal domains may become increasingly important under prothrombotic conditions.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

Effect of an anti-sulfatide single-chain antibody probe on platelet function

2008 ◽  
Vol 99 (03) ◽  
pp. 552-557 ◽  
Author(s):  
Corie Shrimpton ◽  
Koichi Honke ◽  
Rolando Rumbaut ◽  
Jose Lopez ◽  
Perumal Thiagarajan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Lag Phase ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Platelet Surface ◽  
Occlusion Time ◽  
Single Chain Fragment Variable

SummarySulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis.We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 μg/ml) inhibited the adhesion of activated platelets to fibrinogen,VWF, P-selectin,TSP1 and laminin by 30%, 30%,75%,20% and 35%,respectively,compared to a control scFv (p<0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p<0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p<0.05).Under flow PA38 decreased the thrombus formation on collagen by 31%, (p<0.01). Sulfatidedeficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p<0.05), though in-vivo haemostasis did not differ significantly.Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.

Modulation of von Willebrand Factor A1 Domain-Mediated Platelet Aggregation/Agglutination by α-Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 628-628
Author(s):  
Grazia Loredana Mendolicchio ◽  
Reha Celikel ◽  
Kottayil I. Varughese ◽  
Brian Savage ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Amino Terminal ◽  
Low Responder ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Evaluation of the crystal structures of the amino terminal domain of platelet glycoprotein (GP) Ibα bound to the von Willebrand factor A1 domain (VWFA1) or to α-thrombin indicate the absence of significant steric hindrance in a putative triple complex of the two ligands interacting with the same receptor molecule. Superposition of the models reveals that intermolecular contacts may be established between VWFA1 and α-thrombin concurrently bound to GP Ibα, and suggests that these additional interactions could stabilize the intrinsically low affinity binding of the VWF A1 domain. To verify the predictions of the model, we used gel electrophoresis under native conditions and purified components in solution to demonstrate directly the formation of a triple complex. We then sought to evaluate whether α-thrombin could influence the functional effects of the VWF-GP Ibα interaction. For this purpose, we established a model of platelet agglutination/aggregation dependent on the interaction between recombinant dimeric VWFA1 domain, purified from the culture medium of stably transfected D. melanogaster cell lines, and GP Ibα. In this assay, platelet rich plasma prepared from individual donor blood collected with the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK) as an anticoagulant (80 μM) was mixed with varying concentrations of dimeric VWFA1 (0.5-10 μg/ml) and exposed to variable shear rate levels in a cone-and-plate viscometer. Platelet aggregation was observed at shear rates between 6 and 108 dyn/cm2. The response in different normal controls was reproducible but variable in extent, and individuals could be assigned to one of two categories, low responder and high responder. An agglutination response was observed after platelets were treated with 10 μM prostaglandin E1 to block activation, and the distinction between low and high responders remained true under these conditions. For simplicity, agglutinated platelets were still defined as “aggregates”. With activation blocked platelets, aggregates were stable up to a shear rate of 30 dyn/cm2, but began to dissipate at higher levels. The addition of α-thrombin with the active site irreversibly blocked by PPACK at concentrations between 5 and 10 μg/ml substantially increased the extent of the platelet response. This was demonstrated by a faster rate of platelet agglutination/aggregation, a greater stability of aggregates at higher shear rates, and an overall increase in the size of aggregates formed. To demonstrate the latter, samples were exposed to shear stress under selected conditions and immediately fixed with 1% glutaraldehyde for quantitative image analysis. Maximum aggregate size was increased several fold in the presence of α-thrombin, and the difference was particularly evident in low responder individuals in whom dimeric VWFA1 alone caused the formation of small and unstable aggregates. PPACK-blocked thrombin by itself had no effect on platelet aggregate formation at any shear rate tested. Our findings delineate a mechanism through which α-thrombin may stabilize platelet-platelet contacts by mediating a tighter association between VWF A1 domain and GP Ibα receptor. Such a function, independent of proteolytic activity, may enhance platelet deposition at sites of vascular injury.

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