Protein Tyrosine Phosphorylation in Human Platelets during Shear Stress-Induced Platelet Aggregation (SIPA) Is Regulated by Glycoprotein (GP) Ib/IX as well as GP IIb/IIIa and Requires Intact Cytoskeleton and Endogenous ADP

1995 ◽  
Vol 74 (02) ◽  
pp. 736-742 ◽  
Author(s):  
Atsushi Oda ◽  
Kenji Yokoyama ◽  
Mitsuru Murata ◽  
Michihide Tokuhira ◽  
Kosei Nakamura ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Phosphorylated Proteins ◽  
Sds Page ◽  
Stenotic Arteries ◽  
Von Willebrand

SummaryShear stress-induced platelet aggregation (SIPA) may be essential in thrombus formation in pathologically stenotic arteries. Intracellular events during SIPA are, however, poorly understood. Washed platelets were exposed to shear stress (108 dyne/cm2) in the presence of von Willebrand factor (vWf, 10 μg/ml) and 1 mM CaCl2 for various time intervals, and then lyzed in SDS. Platelet proteins were separated by 10% SDS-PAGE and tyrosine phosphorylated proteins were detected by immunoblotting with an anti-phosphotyrosine monoclonal antibody. Increased tyrosine phosphorylation of proteins of 130, 100, 85, 74, 70, 64, 58, and 40 kDa was observed within 30 s after the beginning of exposure of platelets to high shear force and the degree of tyrosine phosphorylation continued to increase up to approximately 2 min after the exposure. A monoclonal antibody (MoAb) against vWf-binding domain of glycoprotein (GP) Ibα (GUR83-35), anti-vWf MoAb that inhibits binding of vWf to GPIbα (NMC-4), or a MoAb against GP IIb/IIIa complex (AP-2) inhibited SIPA as well as tyrosine phosphorylation of these proteins. Apyrase (an ADP scavenger, 2 U/ml), EDTA (5 mM), or RGDS peptide (200 μg/ml) also had inhibitory effects on both SIPA and tyrosine phosphorylation. However, Cytochalasin D (2 μM) or staurosporin (1 μM) did not affect SIPA, while they inhibited SIPA-associated tyrosine phosphorylation of those proteins. SIPA-associated tyrosine phosphorylation is a novel post-aggregatory pathway in signal transduction, which is dependent on the binding of vWf to GP Ib/IX and GP IIb/IIIa, endogenous ADP, and intact cytoskeleton.

STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 157-157
Author(s):  
Zhou Zhou ◽  
Francisca C. Gushiken ◽  
Angela Bergeron ◽  
Vinod K. Vijayan ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcription Factor ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Dose Dependent

Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
Author(s):  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Paola Pradella ◽  
Adriana Masotti ◽  
Francesco I Pareti
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Transmembrane Flux ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

scholarly journals cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Stephanie Makhoul ◽  
Katharina Trabold ◽  
Stepan Gambaryan ◽  
Stefan Tenzer ◽  
Daniele Pillitteri ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Light Transmission ◽  
Thrombus Formation ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Induce Platelet Aggregation ◽  
Akt Phosphorylation ◽  
Camp And Cgmp

Abstract Background The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. Methods Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. Results EB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin−/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. Conclusion EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα−/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα. Graphical abstract

scholarly journals Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
JL Moake ◽  
NA Turner ◽  
NA Stathopoulos ◽  
L Nolasco ◽  
JD Hellums
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Fluid Shear Stress ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Shear Stresses ◽  
Fluid Shear ◽  
Von Willebrand ◽  
Induced Aggregation

Abstract Fluid shear stress in arteries and arterioles partially obstructed by atherosclerosis or spasm may exceed the normal time-average level of 20 dyne/cm2. In vitro, at fluid shear stresses of 30 to 60 dyne/cm2 applied for 30 seconds, platelet aggregation occurs. At these shear stresses, either large or unusually large von Willebrand factor (vWF) multimers in the suspending fluid exogenous to the platelets mediates aggregation. Adenosine diphosphate (ADP) is also required and, in these experiments, was released from the platelets subjected to shear stress. At 120 dyne/cm2, the release of endogenous platelet vWF multimers can substitute for exogenous large or unusually large vWF forms in mediating aggregation. Endogenous released platelet vWF forms, as well as exogenous large or unusually large vWF multimers, must bind to both glycoproteins Ib and the IIb/IIIa complex to produce aggregation. Shear- induced aggregation is the result of shear stress alteration of platelet surfaces, rather than of shear effects on vWF multimers. It is mediated by either large plasma-type vWF multimers, endogenous released platelet vWF forms, or unusually large vWF multimers derived from endothelial cells, requires ADP, and is not inhibited significantly by aspirin. This type of aggregation may be important in platelet thrombus formation within narrowed arterial vessels, and may explain the limited therapeutic utility of aspirin in arterial thrombosis.

scholarly journals Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 113-120 ◽  
Author(s):  
TW Chow ◽  
JD Hellums ◽  
JL Moake ◽  
MH Kroll
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

scholarly journals Shear-stress-induced von Willebrand factor binding to platelets causes the activation of tyrosine kinase(s)

1994 ◽  
Vol 302 (3) ◽  
pp. 681-686 ◽  
Author(s):  
K Razdan ◽  
J D Hellums ◽  
M H Kroll
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Von Willebrand Factor ◽  
Arterial Blood Flow ◽  
Shear Stresses ◽  
Arterial Blood ◽  
Stress Dependent ◽  
Von Willebrand ◽  
Willebrand Factor

Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca2+ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29,000-31,000, 36,000, 50,000, 58,000, 64,000, 76,000, 85,000 and 105,000 was observed. PTP in response to a threshold shear stress of 0.3 mN/cm2 (30 dyn/cm2) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm2 (90 dyn/cm2) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca2+ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r) approximately 31,000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r) approximately 29,000 was shear-stress-dependent but independent of extracellular Ca2+. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r) approximately 76,000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.

scholarly journals Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 113-120 ◽  
Author(s):  
TW Chow ◽  
JD Hellums ◽  
JL Moake ◽  
MH Kroll
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Abstract Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

scholarly journals Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
JL Moake ◽  
NA Turner ◽  
NA Stathopoulos ◽  
L Nolasco ◽  
JD Hellums
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Fluid Shear Stress ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Shear Stresses ◽  
Fluid Shear ◽  
Von Willebrand ◽  
Induced Aggregation

Fluid shear stress in arteries and arterioles partially obstructed by atherosclerosis or spasm may exceed the normal time-average level of 20 dyne/cm2. In vitro, at fluid shear stresses of 30 to 60 dyne/cm2 applied for 30 seconds, platelet aggregation occurs. At these shear stresses, either large or unusually large von Willebrand factor (vWF) multimers in the suspending fluid exogenous to the platelets mediates aggregation. Adenosine diphosphate (ADP) is also required and, in these experiments, was released from the platelets subjected to shear stress. At 120 dyne/cm2, the release of endogenous platelet vWF multimers can substitute for exogenous large or unusually large vWF forms in mediating aggregation. Endogenous released platelet vWF forms, as well as exogenous large or unusually large vWF multimers, must bind to both glycoproteins Ib and the IIb/IIIa complex to produce aggregation. Shear- induced aggregation is the result of shear stress alteration of platelet surfaces, rather than of shear effects on vWF multimers. It is mediated by either large plasma-type vWF multimers, endogenous released platelet vWF forms, or unusually large vWF multimers derived from endothelial cells, requires ADP, and is not inhibited significantly by aspirin. This type of aggregation may be important in platelet thrombus formation within narrowed arterial vessels, and may explain the limited therapeutic utility of aspirin in arterial thrombosis.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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