Thrombin decreases von Willebrand factor binding to platelet glycoprotein Ib

Thrombin is a physiological agonist that promotes platelet aggregation and secretion. In this study we observed that thrombin can also inhibit a function of platelets related to primary hemostasis. Platelet stimulation by thrombin decreased the binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib and decreased ristocetin-induced agglutination, in vitro reactions that correlate with initial platelet adhesion to the vessel wall. Binding of the monoclonal antibody API to GP Ib was also decreased. Cytoskeletal participation in the change of GP Ib was suggested because pretreatment of platelets with cytochalasin to prevent actin filament formation prevented the thrombin-induced decreases in vWF binding. API binding, and ristocetin-induced agglutination. Measurement of GP Ib in detergent extracts by electroimmunoassay demonstrated no loss after thrombin stimulation. Electroimmunoassay also demonstrated that the API epitope of GP Ib on intact thrombin-treated platelets was accessible for complete digestion by chymotrypsin. Therefore GP Ib was neither released from the platelet surface nor internalized by thrombin treatment. A previously recognized effect of thrombin is its induction of receptor sites on platelet surface GP IIb-IIIa for contact-promoting proteins, including vWF that are involved in the platelet spreading and aggregation that follow adhesion. Therefore the action on GP Ib may combine with the effect on GP IIb-IIIa to shift platelet reactivity from GP Ib-vWF-mediated initial contact with the vessel wall to GP IIb-IIIa-mediated spreading and aggregation.     

Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies

Two murine monoclonal antibodies, FMC 25 and AN 51, directed against distinct epitopes on the glycoprotein Ib complex, have been used to further define the mechanism of quinine/quinidine drug-dependent antibody interaction with platelets. FMC 25, directed against an epitope on glycoprotein IX, had no effect on platelet aggregation induced by collagen or adenosine diphosphate and little, if any, effect on ristocetin-induced platelet agglutination. FMC 25 and its (Fab)2 fragment, however, were potent inhibitors of drug-dependent antibody- induced platelet aggregation and blocked binding of drug-dependent antibody to platelets as assessed by indirect platelet immunofluorescence. In contrast, AN 51, directed against an epitope on the alpha-subunit of glycoprotein Ib, blocked ristocetin-induced, factor VIII/von Willebrand factor (FVIII/vWF)-dependent platelet agglutination but not drug-dependent antibody-induced platelet aggregation or binding of drug-dependent antibody to platelets. Selective proteolytic removal of the majority of the alpha-subunit of glycoprotein Ib (glycocalicin) from platelets by treatment with calcium- dependent protease did not affect binding of drug-dependent antibody. In addition, a quinidine-dependent antiplatelet antibody immunoprecipitated glycoprotein Ib complex from normal platelets and the membrane-associated proteolytic remnant of the glycoprotein Ib complex from calcium-dependent protease-treated platelets. Preincubation of drug-dependent antibody with purified glycoprotein Ib complex inhibited subsequent binding of antibody to platelets, but the separated components, glycoprotein Ib and glycoprotein IX, were both ineffective, suggesting that the normal interaction between glycoprotein Ib and glycoprotein IX in the intact complex was necessary for drug-dependent antibody recognition. The functional response of platelets to drug-dependent antibody was not mediated by way of platelet Fc receptor, since aggregation of washed platelets by acetone- aggregated IgG was not inhibited by FMC 25 (Fab)2. FVIII/vWF was not required for drug-dependent antibody-induced platelet aggregation. The combined evidence is consistent with quinine/quinidine-dependent antibody-platelet interaction occurring by way of a FVIII/vWF- independent, Fc receptor-independent mechanism that probably involves binding of antibody to glycoprotein IX or the beta-subunit of glycoprotein Ib or both.     

Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function

Summary. Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01–0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with <0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min. indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31±0.90 for resting platelets, down-regulated to 0.84±0.13 ( P >0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63±2.21 ( P >0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.     

The mechanism of heparin-induced platelet aggregation

When heparin was added to platelet-rich plasma, mild but irreversible platelet aggregation was demonstrated. This platelet response was not accompanied by release of alpha-granules and dense body constituents, nor by prostaglandin biosynthesis. It did, however, require metabolic energy and divalent cations as metabolic inhibitors (anti-mycin A and 6-deoxyglucose) and EDTA blocked the reaction. Bernard-Soulier syndrome platelets, which lack glycoprotein (GP) Ib, but not Glanzmann's Thrombasthenia platelets, which lack GP IIb/IIIa, were aggregated by heparin. Monoclonal antibody (mAb) against GP IIb/IIIa, but not mAb against GP Ib, strongly inhibited the reaction. These combined results suggest the participation of GP IIb/IIIa but not GP Ib in heparin-induced platelet aggregation. Fibrinogen was a cofactor in the reaction as gel-filtered platelets were unreactive to heparin but addition of fibrinogen restored their reactivity. Antithrombin III and fibronectin inhibited platelet response to heparin, suggesting that these proteins may protect platelets from aggregation by heparin.     

Studies on a patient with thrombocytopenia, giant platelets and a platelet membrane glycoprotein Ib with reduced amount of sialic acid

A 70-year-old patient with a life-long bleeding tendency, giant platelets and thrombocytopenia (10-40 x 10(9) platelets/l) has been studied. This is a condition often associated with lack of platelet membrane glycoprotein Ib (GP Ib). Electron microscopy of fixed platelets incubated with monoclonal antibodies to GP Ib (AN 51, AP 1) and gold-labelled goat anti-mouse IgG, showed a distinct distribution of GP Ib on the patient's platelets, however. Crossed immunoelectrophoresis and SDS-PAGE demonstrated a reduced mobility of the patient's GP Ib which could be explained by absence of sialic acid. Blotting with peroxidase-conjugated peanut agglutinin confirmed this conclusion. This lectin binds to galactose-N-acetyl-galactosamine residues exposed terminally when sialic acid is absent from the carbohydrate side-chains. Such binding could be seen with normal GP Ib only after neuraminidase treatment. Fluorescence studies with FITC-conjugated peanut agglutinin showed binding of the lectin to intact patient platelets, indicating that lack of sialic acid was not introduced during the platelet isolation procedure. Neither could the lack of sialic acid be attributed to increased neuraminidase activity as studied in vitro. Platelets treated with neuraminidase in vivo or in vitro are rapidly cleared from the circulation. Therefore the patient's thrombocytopenia may be associated with the reduced amount of GP Ib sialic acid. As far as we know, similar cases have not been described previously.     

Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.     

Molecular characterization of two mutations in platelet glycoprotein (GP) Ib alpha in two Finnish Bernard-Soulier syndrome families

Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder and macrothrombocytopenia which is caused by a defect in the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the receptor for von Willebrand factor and thrombin. Here we report the molecular basis of the classical form of BSS in two unrelated Finnish patients, both with a life-long history of severe bleeding. Flow cytometry and immunoblotting showed no expression of GP Ib/IX, GP Ib alpha, GP Ib beta or GP IX (less than 10%) in the patients' platelets. No expression of GP V (< 10%) was observed in propositus 1, but a residual amount was found in propositus 2 (24%). DNA sequencing analysis revealed that propositus 1 was compound heterozygous for a two-base-pair deletion at Tyr505(TAT) and a point mutation Leu129(CTC)Pro(CCC) in the GP Ib alpha gene. Propositus 2 was homozygous for the Tyr505(TAT) deletion. The nine relatives who were heterozygous for either of the mutations also had low levels of GP Ib alpha (74-90%). Hence, Bernard-Soulier patients homozygous or compound heterozygous for Tyr505(TAT) are severely affected. Interestingly, both mutations have independently been found in three other families in previous reports, suggesting their ancient age or mutational 'hot spot'.     

Stability of Glycoproteins Ib/IX and IIb/IIIa during Preparation and Storage of Platelet Concentrates: Detection by Binding Assays with Epitope-Defined Monoclonal Antibodies and Physiological Ligands

Preservation of the glycoprotein (GP) complexes Ib/IX and IIb/IIIa, because of their role as specific platelet receptors for adhesive proteins, is essential for the haemostatic efficacy of transfusions of platelet concentrates (PCs). We have combined binding assays with epitope-defined monoclonal antibodies, and with the physiological ligands von Willebrand factor and fibrinogen (Fo), to investigate the total expression and functional status of these receptors, in PCs stored for up to 9 days. Routine preparation and storage for up to 5 days have no apparent effect on the surface expression and the functional status of GPs Ib/IX and IIb/IIIa. However, after prolonged storage for up to 9 days we found a 50% decrease in the level of the total and functional GP Ib/IX content of platelets. This finding parallelled a significant rise in plasma glycocalicin, a proteolytic fragment of GP Ib. In addition, long-term storage promoted an impairment in the exposure of GP IIb/IIIa to Fo, and a 50% decrease in Fo binding capacity, without affecting the complex quantiatitvely. Finally, we also noticed a storage-induced increase in the platelet surface expression of GMP 140, and after 9 days of storage there was a rise in mean platelet volume, and a significant reduction in pH levels.     

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.     

Shear-dependent functions of the interaction between soluble von Willebrand factor and platelet glycoprotein Ib in mural thrombus formation on a collagen surface

Recent flow studies have clearly established the function of von Willebrand factor (vWF) in initial platelet adhesion, in which the interaction of surface-immobilized vWF with platelet glycoprotein (GP) Ib, particularly at high shear rates, leads to the transient capture of flowing platelets onto the surface. This interaction is thought to trigger activation of platelet GP IIb/IIIa, leading to irreversible platelet adhesion and subsequent mural thrombus growth. The role of vWF-GP Ib interaction in secondary thrombus growth remains to be clarified, however. In this study, time-course images of the thrombus formation process were obtained using a whole blood flow system that allows real-time visualization of fluorescence-labeled platelet thrombus formation. The system employs a collagen-coated surface in a parallel plate flow chamber mounted on an epifluorescence microscope, which is then subjected to computer-assisted image analysis. In perfusion of blood preincubated with anti-vWF antibody NMC-4, which blocks the vWF-GP Ib interaction in solution, neither primary platelet adhesion nor subsequent thrombus growth on a collagen surface was detected at high shear rates (> or = 1210/s). In addition, even under experimental conditions in which initial platelet adhesion normally occurred, NMC-4-treated blood perfusion showed an apparent defect of secondary thrombus growth at the same high shear rates. The overall process of thrombus formation at low shear rates (< or = 340/s) was not affected by specific blockers of the vWF-GP Ib interaction. These findings indicate that, in addition to the interaction of surface-immobilized vWF with GP Ib in platelet adhesion, the interaction of soluble vWF with GP Ib is required for secondary thrombus growth selectively at high shear rates.     

Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test

The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one- stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP- induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.     

Glycoprotein V is not the thrombin activation receptor on human blood platelets

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.     

Role and initiation mechanism of the interaction of glycoprotein Ib with surface-immobilized von Willebrand factor in a solid-phase platelet cohesion process

To know the role and initiation mechanism of the interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF), we examined the effect of shear stress levels on platelet binding to vWF-coated plates using a cone-and-plate type viscometer capable of loading various levels of shear stress. The extent of platelet binding to immobilized vWF reached a plateau at the shortest period tested (20 seconds) under high shear stress (90 dyne/cm2), whereas 9 to 12 minutes was necessary for saturable platelet binding under static conditions. This shear effect, which was found to be dependent on the vWF-GP Ib interaction, was observed even under the lowest shear stress (1.5 dyne/cm2) examined. In contrast with the high shear effect previously reported to initiate the interaction of GP Ib with soluble vWF, these results indicate that relatively low levels of shear stress can promote the interaction of GP Ib with surface- immobilized vWF. This effect of shear stress was observed regardless of the manner in which vWF was immobilized, suggesting that immobilization itself and not, as previously hypothesized, a conformational change in vWF induced by direct adsorption to the surface is responsible for the enhanced GPIb binding. Thus, the present findings suggest that the vWF- GP Ib interaction contributes optimally to rapid platelet cohesion on a thrombogenic surface when vWF is in a static state and when platelets are moved by an appropriate rheological force such as low shear stress.     

Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC- 1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA- fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb- IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.     

Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3).

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin alpha(IIb)beta(3). GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin alpha(IIb)beta(3) promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin alpha(IIb)beta(3), particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3-kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3-kinase lipid product, PtdIns(3,4)P(2). Under static conditions, inhibiting PI 3-kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin alpha(IIb)beta(3) activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3-kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3-kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3-kinase dramatically reduced intracellular calcium mobilization and integrin alpha(IIb)beta(3) activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3-kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3-kinase-dependent and -independent mechanisms, whereas under shear PI 3-kinase is indispensable for VWF-induced calcium release.     

Glycoprotein Ib-V-IX, a receptor for von Willebrand factor, couples physically and functionally to the Fc receptor gamma-chain, Fyn, and Lyn to activate human platelets.

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin alpha(IIb)beta(3). We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (triangle upA1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor gamma-chain through interaction with Syk SH2 domains. GP Ib physically associates with the gamma-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cgamma2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated gamma-chain, phosphorylation of PLCgamma2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR gamma-chain and members of the Src family kinases, leading to phosphorylation of the gamma-chain, recruitment, and activation of Syk. Phosphorylation of PLCgamma2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.     

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)