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M R Barnard

Publications and source records attributed to M R Barnard.

At least 19 recordsLinked to original sources
PubMed1996

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Animals↗
PubMed1996

Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.

Highly reactive oxygen species rapidly inactivate nitric oxide (NO), and endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients'platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H2O2 generation in one patient and a 3.4-fold increase in the second patient, both compared woth control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.

Cyclic GMP↗
PubMed1996

The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system.

In this study, we show that the platelet surface expression of glycoprotein (GP) V is regulated by two independent mechanisms. While confirming that both thrombin and neutrophil elastase proteolyse GPV, we show that neutrophil cathepsin G, thrombin receptor activating peptide (TRAP), and a combination of ADP and epinephrine can each result in a decrease in the platelet surface expression of GPV by a nonproteolytic mechanism: a cytoskeletal-mediated redistribution of platelet surface GPV to the surface-connected canalicular system (SCCS). Four independent lines of evidence documented the nonproteolytic nature of this decrease in the platelet surface expression of GPV. First, flow cytometric studies showed that cathepsin G, TRAP, and ADP/epinephrine decreased the platelet surface expression of GPV without changing the total platelet content of GPV. Second, immunoelectron microscopy directly demonstrated translocation of GPV from the platelet surface to the SCCS. Third, the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV were fully reversible. Fourth, cytochalasin B, an inhibitor of actin polymerization, completely inhibited the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV. The cytoskeletal-mediated redistribution of GPV occurred in a whole blood milieu and at physiologic temperatures (37 degrees C) and extracellular calcium concentrations (2 mmol/L). This study also defines the diverse effects on GPV, GPIb, and GPIX of multiple important platelet agonists. Cathepsin G proteolysed platelet surface GPIb alpha, but redistributed platelet surface GPIX and GPV to the SCCS. Thrombin proteolysed platelet surface GPV, but redistributed platelet surface GPIb and GPIX to the SCCS. Both TRAP and ADP/epinephrine redistributed platelet surface GPIb, GPIX, and GPV to the SCCS. Elastase proteolysed platelet surface GPIb alpha and GPV, but, unlike the other agonists tested, neither proteolysed nor redistributed platelet surface GPIX. The experiments with TRAP showed that activation of the seven-transmembrane domain thrombin receptor can result in translocation of GPIb, GPIX, and GPV to the SCCS independently of the GPIb-mediated pathway of thrombin-induced platelet activation. This study also provides two additional lines of support for the recent report that GPV is noncovalently complexed with GPIb and GPIX in the platelet surface membrane. First, although only the GPIb alpha subunit of this putative complex is known to be directly linked to the platelet cytoskeleton via actin-binding protein, cytochalasin B inhibited the ADP/epinephrine-, cathepsin G-, and TRAP-induced decrease in platelet surface GPV. Second, triple labeling flow cytometric experiments showed that, on each individual platelet, the ADP/epinephrine-induced decrease and subsequent return of the platelet surface expression of GPV occurred simultaneously with the decrease and subsequent return of the platelet surface expression of GPIb. In summary, the platelet surface expression of GPV is regulated by two independent mechanisms: proteolysis and a reversible, cytoskeletal-mediated redistribution to the SCCS.

Actins↗
PubMed1996

Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa.

Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes-Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb-IIIa], AK4 (Mab blocking P-selectin, 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb-IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen receptor). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation.

Amino Acid Sequence↗
PubMed1994

Neutrophil cathepsin G modulates the platelet surface expression of the glycoprotein (GP) Ib-IX complex by proteolysis of the von Willebrand factor binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex.

The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb-IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin-induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed against the macroglycopeptide portion of GPIb alpha). In contrast to its proteolytic effect on GPIb alpha, the cathepsin G-induced decrease in platelet surface GPIX and the remnant of the GPIb-IX complex (defined by MoAbs FMC25 and AK1) was via a cytoskeletal-mediated redistribution, as determined by lack of change in the total platelet content of GPIX and the GPIb-IX complex; complete inhibition by cytochalasin B, prostaglandin I2, and prior fixation of platelets. Experiments with Serratia protease-treated and Bernard-Soulier platelets showed that neither platelet surface GPIb nor cathepsin G-induced proteolysis of GPIb were required for the cathepsin G-induced redistribution of the remnant of the GPIb-IX complex or the cathepsin G-induced increase in platelet surface P-selectin. In summary, neutrophil cathepsin G modulates the platelet surface expression of the GPIb-IX complex both by proteolysis of the vWF binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex. Prior studies show that, although thrombospondin 1, antiserine proteases, and plasma are all inhibitors of cathepsin G, the effects of cathepsin G on platelets, including an increase in surface GPIIb-IIIa, occur during close contact between neutrophils and platelets in a protective microenvironment (eg, thrombosis and local inflammation).(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1994

The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible.

Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time-dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb-IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation-induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

Adult↗
PubMed1994

Human neutrophil cathepsin G is a potent platelet activator.

PURPOSE: Neutrophil activation has been implicated in the pathophysiologic condition of ischemia-reperfusion injury, the formation of arterial aneurysms, the progression of myocardial ischemia, and the initiation of deep venous thrombosis. Activated neutrophils release cathepsin G, a serine protease, from their granules, which may cause platelet activation that leads to intravascular thrombosis, tissue infarction, and systemic release of the thrombogenic products of platelet granules. This study used flow cytometry to quantify the extent of cathepsin G-induced platelet activation and degranulation through changes in the expression of platelet surface glycoproteins. METHODS: Increasing concentrations of human neutrophil-derived cathepsin G were incubated with washed platelets or whole blood from healthy human donors. The platelet surface expression of glycoproteins, including P-selectin, a platelet membrane glycoprotein only expressed after platelet alpha granule release, were determined by quantifying the platelet binding of a panel of fluorescently labeled monoclonal antibodies. Results were compared with the effect of a maximal dose of thrombin, the most potent known platelet activator. RESULTS: In a washed platelet system, cathepsin G increased platelet surface expression of P-selectin (an activation-dependent neutrophil binding site), the glycoprotein IIb/IIIa complex (fibrinogen receptor), and glycoprotein IV (thrombospondin receptor), and decreased surface expression of glycoprotein Ib (von Willebrand factor receptor) to an extent comparable to maximal thrombin. However, these effects were not observed in a whole blood system. Further experiments revealed that preexposure to plasma completely inhibited cathepsin G-induced washed platelet activation and degranulation. Prostacyclin treatment of washed platelets markedly inhibited cathepsin G-induced platelet activation. CONCLUSIONS: Cathepsin G is a very potent platelet agonist and degranulator, comparable to maximal thrombin, which alters platelet surface glycoprotein expression for enhanced neutrophil binding and effective platelet aggregation. This study helps to elucidate a possible pathway through which neutrophils may directly activate platelets, leading to intravascular thrombosis, irreversible ischemia, and tissue death in cardiovascular disease states. Patients with diseased endothelium that is deficient in prostacyclin production may be particularly prone to the detrimental effects of neutrophil-derived cathepsin G platelet activation.

Antigens, CD↗
PubMed1994

Platelet activation results in a redistribution of glycoprotein IV (CD36).

To investigate the possibility that thrombin and/or other platelet activators change the platelet surface expression of glycoprotein IV (GPIV, CD36), we used a panel of five GPIV-specific monoclonal antibodies (OKM5, 5F1, FA6-152, 8A6, and F13) directed against different epitopes. All these antibodies bound to resting platelets in a concentration-dependent and saturable manner, as determined by flow cytometry of washed platelets. Thrombin (1 U/mL) induced an approximately twofold increase in the platelet surface binding of each of these monoclonal antibodies. Immunofluorescence microscopy demonstrated an internal pool of GPIV that, after thrombin stimulation, redistributed to the platelet surface. In a whole-blood flow-cytometric assay, alpha-thrombin and the thromboxane A2 analogue U46619 each resulted in an approximately twofold increase in the platelet surface binding of OKM5, whereas ADP had a more modest effect, and collagen and epinephrine had little effect. The activation-induced up-regulation of the platelet OKM5 epitope occurred in vivo as demonstrated by flow cytometric analysis of whole blood emerging from a standardized skin puncture site. In summary, both in vitro and in vivo platelet activation results in increased platelet surface expression of GPIV, as a result of a redistribution of GPIV from an internal pool.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Reversible inhibition of human platelet activation by hypothermia in vivo and in vitro.

A hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22 degrees C and 37 degrees C. the forearm skin temperature was maintained at temperatures between 22 degrees C and 37 degrees C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB2 (the stable metabolite of TXA2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37 degrees C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB2 generation, and the bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood.

Previous studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human alpha-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex.(ABSTRACT TRUNCATED AT 250 WORDS)

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Effect of strenuous exercise on platelet activation state and reactivity.

BACKGROUND: It has been hypothesized that platelets are activated, or made more activatible, by strenuous exercise and that these changes may play a role in the genesis of exercise-induced coronary ischemia. Previous studies have yielded conflicting results but have used assays (eg, platelet aggregation, plasma platelet factor 4, and plasma beta-thromboglobulin) that are subject to methodological problems. METHODS AND RESULTS: In the present study, a whole blood flow cytometric method was used to study the platelet activation state and reactivity of 12 physically active and 12 sedentary individuals before and after standardized treadmill exercise testing. The peptide gly-pro-arg-pro (GPRP) was included in this assay to prevent fibrin polymerization and platelet aggregation, thus allowing the measurement of the reactivity to thrombin of individual platelets in the physiological milieu of whole blood. A panel of fluorescent-labeled monoclonal antibodies was used to monitor activation-dependent platelet surface changes: downregulation of glycoprotein (GP) Ib (6D1) and upregulation of GMP-140 (S12), the GPIIb-IIIa complex (PAC1), and GPIV (OKM5). In samples obtained before exercise, platelets not exposed to thrombin showed no evidence of in vitro activation. In the sedentary subjects, exercise caused a consistent and significant augmentation of the platelet activation state and reactivity as judged by the binding of 6D1 in the presence of thrombin 0.05 U/mL (P < .001), 0.005 U/mL (P = .001), and 0 U/mL (P = .004) and by the binding of OKM5 in the presence of thrombin 0.05 U/mL (P < .001), 0.005 U/mL (P = .029), and 0 U/mL (P = .035). Exercise increased the binding of PAC1 at only a single thrombin concentration (0.005 U/mL, P = .027) and did not alter the binding of S12 at any thrombin concentration. In contrast, in the physically active subjects, exercise failed to cause a consistent alteration in either platelet activation state or platelet reactivity. No significant differences were found between the 12 male and 12 female volunteers. CONCLUSIONS: Strenuous exercise in sedentary subjects but not physically active subjects resulted in both platelet activation and platelet hyperreactivity. These changes were more readily detected with monoclonal antibodies directed against GPIb (6D1) and, to a lesser extent, GPIV (OKM5) rather than those directed against the GPIIb-IIIa complex (PAC1) and GMP-140 (S12). Platelet activation by thrombin, generally regarded as the most physiologically important agonist, can be studied in whole blood in a clinical setting through the use of the peptide GPRP.

Adult↗
PubMed1992

High-dose heparin suppresses platelet alpha granule secretion.

Platelet degranulation has been implicated in the pathophysiology of acute arterial thrombosis, intimal hyperplasia, and atherogenesis. Most previous studies that examined the effect of heparin on platelet function have used platelet aggregometry. These studies have resulted in contradictory data and, by the nature of the assay, reveal no information with regard to platelet degranulation. In contrast, flow cytometry allows accurate quantification of the extent of platelet degranulation by measurement of the platelet surface binding of a GMP-140 specific monoclonal antibody (S12). GMP-140 is only expressed on the platelet surface after platelet alpha granule release. In the present study increasing concentrations of heparin were added to whole blood anticoagulated with sodium citrate. Platelets were activated with a panel of agonists, and the extent of platelet degranulation was quantified by whole blood flow cytometry. Heparin concentrations as high as 100 units/ml were found to suppress platelet alpha granule release induced by either a thromboxane A2 analog (U46619) or a combination of adenosine diphosphate and epinephrine. Heparin suppressed alpha granule release induced by thrombin both in whole blood and in washed platelets. The addition of heparin after platelet activation had no effect on S12 binding. In summary, heparin in high concentrations is a potent inhibitor of platelet degranulation, an action that is unrelated to its effect on the coagulation cascade. Although the heparin concentrations used in this study exceed those used clinically by a factor of 10 or more, future studies of heparin fractions may allow the separation of the anticoagulant and antiplatelet properties of the molecule and allow the administration of an agent that selectively suppresses platelet degranulation without the humoral anticoagulant effect.

Adenosine Diphosphate↗
PubMed1991

Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation.

In this study, the question of whether glycoprotein Ib (GPIb) mediates both high and moderate affinity pathways of alpha-thrombin-induced platelet activation was examined. Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP-140 that were induced by high concentrations of alpha-thrombin (10 nmol/L). However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3). When present in 20-fold molar excess, an MoAb directed against the alpha-thrombin/von Willebrand factor (vWf) binding domains of GPIb completely blocked the ristocetin-dependent binding of vWf to platelets but inhibited only to about 50% the binding of alpha-thrombin and the activation-dependent binding of vWf. In platelets treated with Serratia marcescens protease to remove GPIb, a concentration of this MoAb 16,000-fold in excess of the maximum possible remaining copies of GPIb failed to inhibit platelet activation by alpha-thrombin. These studies demonstrate that activation of intact platelets by alpha-thrombin proceeds by both GPIb-dependent and GPIb-independent mechanisms.

Antibodies, Monoclonal↗
PubMed1991

Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound.

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

Actins↗
PubMed1990

Plasmin-induced redistribution of platelet glycoprotein Ib.

Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin-induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).

Blood Platelets↗
PubMed1988

Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

Platelet membrane glycoprotein (GP) Ib contains receptor for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In summary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).

Antibodies, Monoclonal↗