MoAb 7E3 Research Articles

Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of αIIbβ3 engagement

AbstractShear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.

Monoclonal antibodies to αVβ3 (7E3 and LM609) inhibit sickle red blood cell–endothelium interactions induced by platelet-activating factor: Presented in part at the INSERM Symposium, Paris, France, September 2, 1995 (ref 24), and at the 40th Annual Meeting of the American Society of Hematology, Miami, FL, December 7, 1998.

Abnormal interaction of sickle red blood cells (SS RBC) with the vascular endothelium has been implicated as a factor in the initiation of vasoocclusion in sickle cell anemia. Both von Willebrand factor (vWf) and thrombospondin (TSP) play important roles in mediating SS RBC-endothelium interaction and can bind to the endothelium via alphaVbeta3 receptors. We have used monoclonal antibodies (MoAb) directed against alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa) integrins to dissect the role of these integrins in SS RBC adhesion. The murine MoAb 7E3 inhibits both alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa), whereas MoAb LM609 selectively inhibits alphaVbeta3, and MoAb 10E5 binds only to alphaIIbbeta3. In this study, we have tested the capacity of these MoAbs to block platelet-activating factor (PAF)-induced SS RBC adhesion in the ex vivo mesocecum vasculature of the rat. Infusion of washed SS RBC in preparations treated with PAF (200 pg/mL), with or without a control antibody, resulted in extensive adhesion of these cells in venules, accompanied by frequent postcapillary blockage and increased peripheral resistance units (PRU). PAF also caused increased endothelial surface and interendothelial expression of endothelial vWf. Importantly, pretreatment ofthe vasculature with either MoAb 7E3 F(ab')(2) or LM609, but not 10E5 F(ab')(2), after PAF almost completely inhibited SS RBC adhesion in postcapillary venules, the sites of maximal adhesion and frequent blockage. The inhibition of adhesion with 7E3 or LM609 was accompanied by smaller increases in PRU and shorter pressure-flow recovery times. Thus, blockade of alphaVbeta3 may constitute a potential therapeutic approach to prevent SS RBC-endothelium interactions under flow conditions. (Blood. 2000;95:368-374)

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

AbstractShear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab′)2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab′)2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab′)2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab′)2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.

Analysis of GPIIb/IIIa Receptor Number by Quantification of 7E3 Binding to Human Platelets

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab')2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab')2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab')2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab')2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.

Role of Platelets, Thrombin, Integrin llb-llla, Fibronectin and Von Willebrand Factor on Tumor Adhesion in Vitro and Metastasis in Vivo

The role of platelets and thrombin was examined in tumor cell adhesion in vitro and metastasis in vivo. Adhesion of tumor cells to platelets was inhibited by agents inhibiting platelet integrin IIb-IIIa receptor occupancy (MoAb 10E5 and tetrapeptide RGDS) as well as IIb-IIIa ligands (polyclonal antibodies against fibronectin and von Willebrand factor (vWF)). In vivo murine experimental pulmonary metastasis (tail vein injection) could be inhibited by antibody-induced induction of thrombocytopenia and reconstituted by simultaneous injection of human platelets. Preincubation of human platelets with MoAb 10E5 (vs IIb-IIIa) inhibited reconstitution of metastasis. Thrombin activates tumor cell adhesion to platelets by activating platelets as well as tumor cells. Thrombin-activated tumor cells also enhance their adhesion to endothelial cells as well as adhesive ligands fibronectin and vWF. Experimental pulmonary metastasis is enhanced 4-400 fold by preinfusion of thrombin into mice or 10-160 fold by prior treatment of tumor cells with thrombin. The in vitro and in vivo effects of thrombin were mimicked by thrombin receptor activation peptides SFLLRNPNDKYEPF and SFLLRN. Nine of nine tumor cell lines have the seven transmembrane spanning thrombin receptor detected by the polymerase chain reaction. Thus, both platelets and thrombin contribute to experimental tumor metastasis by fostering and enhancing IIb-IIIa integrin platelet interaction with tumor cells. Since many tumor cells generate thrombin, it is proposed that tumor cells may autoactivate a metastatic phenotype.

Prevention of Rethrombosis After Coronary Thrombolysis in a Chronic Canine Model. I. Adjunctive Therapy with Monoclonal Antibody 7E3 F(ab')2 Fragment

We examined the efficacy of the monoclonal antibody (MoAb) 7E3 F(ab')2 fragment, an inhibitor of the platelet glycoprotein (GP)IIb/IIIa receptor, to prevent coronary artery rethrombosis after successful thrombolysis with rt-PA. The circumflex coronary artery of anesthetized dogs was instrumented with a flow probe, an electrode, and a stenosis. After recovery from the surgical procedure, the animals were reanesthetized on post-operative day 9, and vessel wall injury was induced with current applied to the intimal surface of the circumflex coronary artery. The resulting occlusive thrombus was aged for 30 min, and recombinant tissue plasminogen activator (rt-PA) was administered. The animals were allocated to receive either placebo or a single dose of 7E3 [0.8 mg/kg intravenous (i.v.) bolus] as the sole adjunctive agent. Ex vivo platelet function and coronary artery blood flow velocity were recorded on each of 5 consecutive days. Reocclusion and mortality were reduced significantly in animals treated with 7E3 as compared with the placebo-treated group. Significant inhibition of ex vivo platelet aggregation persisted for 48 h after a single injection of 7E3. The MoAb 7E3 F(ab')2 fragment is effective as the sole adjunctive agent with rt-PA for prevention of rethrombosis. The present study is unique in that it examined the efficacy of GPIIb/IIIa inhibition in an experimental model for an extended time, demonstrating the duration of antiplatelet therapy required to prevent rethrombosis after thrombolysis.

Plasmic Degradation of Fibrin Rapidly Decreases Platelet Adhesion and Spreading

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P &gt; .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15–21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P &lt; .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

Prevention of Rethrombosis After Coronary Thrombolysis in a Chronic Canine Model. I. Adjunctive Therapy with Monoclonal Antibody 7E3 F(ab')2 Fragment

Summary We examined the efficacy of the monoclonal antibody (MoAb) 7E3 F(ab')2 fragment, an inhibitor of the platelet glycoprotein (GP)Hb/IIIa receptor, to prevent coronary artery rethrombosis after successful thrombolysis with rt-PA. The circumflex coronary artery of anesthetized dogs was instrumented with a flow probe, an electrode, and a stenosis. After recovery from the surgical procedure, the animals were reanesthetized on postoperative day 9, and vessel wall injury was induced with current applied to the intimal surface of the circumflex coronary artery. The resulting occlusive thrombus was aged for 30 min, and recombinant tissue plasminogen activator (rt-PA) was administered. The animals were allocated to receive either placebo or a single dose of 7E3 [0.8 mg/kg intravenous (i.v.) bolus] as the sole adjunctive agent. Ex vivo platelet function and coronary artery blood flow velocity were recorded on each of 5 consecutive days. Reocclusion and mortality were reduced significantly in animals treated with 7E3 as compared with the placebo-treated group. Significant inhibition of ex vivo platelet aggregation persisted for 48 h after a single injection of 7E3. The MoAb 7E3 F(ab')2 fragment is effective as the sole adjunctive agent with rt-PA for prevention of rethrombosis. The present study is unique in that it examined the efficacy of GPIIb/IIIa inhibition in an experimental model for an extended time, demonstrating the duration of antiplatelet therapy required to prevent rethrombosis after thrombolysis.

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.