Platelet Membrane Glycoprotein Ib Research Articles

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

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.

Shear Stress-Induced von Willebrand Factor Binding to Platelet Glycoprotein lb Initiates Calcium Influx Associated With Aggregation

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.

Platelet Concentrates Stored in Synthetic Medium after Filtration

Platelet concentrates prepared from pooled buffy coats (BCPC) were stored in Plasma-Lyte A, a glucose-free synthetic medium, after leukocyte depletion by filtration through Pall PL 50, and compared to paired unfiltered BCPC stored in the same medium. Each pair of BCPC units was prepared from a pool of 10 buffy coats split into two identical units. Platelet and leukocyte counts per unit of BCPC were 2.70 +/- 0.19 x 10(11) and 3.8 +/- 2.8 x 10(6) (filtered BCPC), 2.59 +/- 0.27 x 10(11) and 79 +/- 56 x 10(6) (control BCPC), respectively. Filtration procedures did not affect in vitro parameters of platelet quality and function such as osmotic reversal, ATP release and aggregation in response to collagen and ADP during 15-day storage. A similar decrease of platelet membrane glycoprotein Ib and a similar rise of activation markers GMP-140, gp 53 and platelet-bound fibrinogen were observed during storage of filtered and control BCPC. Our study indicates that storage of BCPC after filtration is feasible and that a reduction in leukocyte content by filtration to mean cell counts of less than five millions per unit has probably no effect on platelet storage lesion.

Platelet Concentrates Stored in Synthetic Medium after Filtration

Platelet concentrates prepared from pooled buffy coats (BCPC) were stored in Plasma-Lyte A, a glucose-free synthetic medium, after leukocyte depletion by filtration through Pall PL 50, and compared to paired unfiltered BCPC stored in the same medium. Each pair of BCPC units was prepared from a pool of 10 buffy coats split into two indentical units. Platelet and leukocyte counts per unit of BCPC were 2.70 ±0.19 X 10^11 and 3.8 ±2.8x10^6 (filtered BCPC), 2.59 ±0.27 x 10^11 and 79 ±56x10^6 (control BCPC), respectively. Filtration procedures did not affect in vitro parameters of platelet quality and function such as osmotic reversal, ATP release and aggregation in response to collagen and ADP during 15-day storage. A similar decrease of platelet membrane glycoprotein Ib and a similar rise of activation markers GMP-140, gp 53 and platelet-bound fibrinogen were observed during storage of filtered and control BCPC. Our study indicates that storage of BCPC after filtration is feasible and that a reduction in leukocyte content by filtration to mean cell counts of less than five millions per unit has probably no effect on platelet storage lesion.

Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers

Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)- mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF- mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.

Isolation From Commercial Aurintricarboxylic Acid of the Most Effective Polymeric Inhibitors of von Willebrand Factor Interaction With Platelet Glycoprotein Ib. Comparison With Other Polyanionic and Polyaromatic Polymers

Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)-mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF-mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.

Von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb α in platelet-type von Willebrand disease

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib α (GPIb ga), a major component of the platelet (GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI α protein containing residues 228–238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a β-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic ‘face’ consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphiphatic surfaces in the 228–238 region of the Val 233-containing GPIb α protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.

Flow cytometric analysis of anti‐platelet antibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) using acid‐treated, formalin‐fixed platelets

Acid treatment of normal washed platelets prepared by albumin density gradient centrifugation followed by formalin fixation effectively eliminates HLA class I antigens from the platelet surface, without altering the immunoreactivity of platelet membrane glycoproteins Ib and IIb/IIIa complex. Using these platelets, flow cytometric analysis was performed on a total of 20 sera from different patients with chronic ITP, and revealed an inverse linear correlation between the platelet count and the mean fluorescence intensity which represents a relative amount of platelet-bound IgG. These results suggest that acid-treated, formalin-fixed platelets can be used as specimens to detect anti-platelet antibodies to differentiate from those to HLA-class I antigens.

Effects of toluene on platelet membrane glycoprotein Ib and actin-binding protein

Effects of the organic solvent toluene on the platelet membrane receptor glycoprotein Ib (GP Ib) and the cytoskeletal protein, actin-binding protein (ABP), were studied and related to the effects of the local anesthetic dibucaine. The glycocalicin-region of GP Ib contains the binding site for von Willebrand factor; intracellularly GP Ib is linked to the cytoskeleton via ABP. Both GP Ib and ABP are substrates for a calcium-dependent protease, calpain. Washed platelets were incubated with toluene or dibucaine. The toluene concentration in the platelet suspension was analysed by gas chromatography. Using 1.5–2.8 mmol/L toluene, calpain was activated, leading to degradation of ABP and release of glycocalicin from GP Ib. The latter phenomenon was paralleled by a reduced von Willebrand factor-induced platelet agglutination. At lower toluene concentrations (0.3–1.4 mmol/L), degradation of ABP was not detected but an initial increased agglutination that declined to the control level with time was observed. These effects of toluene on the GP Ib-ABP complex are similar to those observed with 1 mmol/L dibucaine. The lowest toluene concentrations used correspond to those that have been found in blood from toluene abusers (“sniffers”).

Similar Effects of Toluene and Dibucaine on Human Platelet Membrane Glycoprotein Ib and Actin‐Binding Protein

Annals of the New York Academy of SciencesVolume 625, Issue 1 p. 835-836 Similar Effects of Toluene and Dibucaine on Human Platelet Membrane Glycoprotein Ib and Actin-Binding Protein ANNE SMITH-KIELLAND, Corresponding Author ANNE SMITH-KIELLAND National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaycAuthor to whom correspondence should be addressed.Search for more papers by this authorANNE MARGRETHE AAKHUS, ANNE MARGRETHE AAKHUS Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this authorÅSE RIPEL, ÅSE RIPEL National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaySearch for more papers by this authorNILS O. SOLUM, NILS O. SOLUM Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this author ANNE SMITH-KIELLAND, Corresponding Author ANNE SMITH-KIELLAND National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaycAuthor to whom correspondence should be addressed.Search for more papers by this authorANNE MARGRETHE AAKHUS, ANNE MARGRETHE AAKHUS Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this authorÅSE RIPEL, ÅSE RIPEL National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaySearch for more papers by this authorNILS O. SOLUM, NILS O. SOLUM Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this author First published: June 1991 https://doi.org/10.1111/j.1749-6632.1991.tb33930.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume625, Issue1Molecular and Cellular Mechanisms of Alcohol and AnestheticsJune 1991Pages 835-836 RelatedInformation

Von Willebrand Factor: Structure and Function

Von Willebrand factor (vWF) is an adhesive, multimeric glycoprotein present in plasma, platelets, and subendothelium, which has two main functions: (1) it serves as a carrier for factor VIII and (2) it plays a crucial role in platelet adhesion to subendothelium, acting as a "bridge" between platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa and subendothelial components such as collagen and heparin. vWF is involved at high shear rates in the initial contact of platelets with the subendothelium, in their subsequent spreading, and in thrombus formation. The three pools of vWF (plasma, platelets, and subendothelium) are necessary for optimal adhesion. Specific fragments of vWF involved in binding to platelets, collagen, heparin, and factor VIII have been mapped by using a series of proteases and well-characterized monoclonal antibodies to distinct epitopes of vWF. Several groups, including ours, have identified at least eight functional domains on the 270-kd subunit that consists of 2,050 amino acids. The importance of the binding domains to GP Ib and to collagen is illustrated by the role of vWF fragment SpIII (amino acids 1 through 1,365) in promoting platelet adhesion to collagen. The role of the vWF-GP Ib axis and of the vWF-GP IIb/IIIa axis in platelet-vessel wall interactions has been demonstrated through the study of patients, monoclonal antibodies, recombinant fragments, and synthetic peptides. We have recently expressed fragments of vWF complementary DNA in Escherichia coli. One of these recombinant fragments, which spans amino acids 449 through 730, binds to platelets in the presence of ristocetin, to collagen, and to heparin and has the property of inhibiting ristocetin-induced platelet agglutination. The second fragment, which spans amino acids 914 through 1,364, binds to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring the release of glycocalicin in platelet concentrates by ELISA

Platelet membrane glycoprotein Ib (GPIb) is one of many GPs on the platelet membrane which contribute to the functional and morphological properties of platelets. A principal function of GPIb is its attachment to von Willebrand Factor (vWF) on injured blood vessels which leads to the adhesion of platelets to these vessels. The binding site to vWF resides on glycocalicin (GC), which is a major segment of GPIb. Glycoprotein Ib is particularly susceptible to centrifugation and storage of platelets. The assessment of GPIb status on platelets, therefore, comprises one of many traditional methods for monitoring the quality of platelets during storage. We have recently developed a novel ELISA to monitor GC levels in the supernatant of platelet concentrates (PCs) during storage. Using this ELISA we observed a progressive rise of GC in PC supernatants during storage. A recent study of citrated PCs with or without EDTA produced similar results, and showed a substantial increase of GC levels in EDTA-treated PCs. The GC ELISA could therefore be used as a novel method to monitor PCs during storage under various conditions.

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

New enzyme-linked immunosorbent assay for glycocalicin in plasma

A new sandwich-type enzyme-linked immunosorbent assay for quantifying glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, is described. The assay is based on the use of two monoclonal antibodies raised against glycoprotein Ib and involves the avidin-biotin technique. The detection limit is 7 micrograms/L and the range of glycocalicin determined in plasma is 0.01 to 1 mg/L. Assay time is 2 h. The intra-assay CV ranged from 3.6% to 5.2%, the interassay CV from 5.4% to 8.0%. Analytical recovery of purified glycocalicin added to a plasma pool averaged 96%. In 36 healthy subjects, the mean glycocalicin concentration in plasma was 0.36 (SD 0.07) mg/L (2.7 nmol/L). We conclude that this assay is suitable for measuring glycocalicin in plasma and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.

Roles of Membrane Glycoproteins on Human Platelet Aggregation Induced by Influenza Virus

The roles of platelet membrane glycoprotein (GP) Ib in human platelet aggregation induced by non-hemolytic influenza virus (IV) were studied. The anti-GPIIIa monoclonal antibody (TM83) which had been shown to inhibit fibrinogen-binding to platelets activated by ADP and thrombin also inhibited the aggregation induced by high and low hemagglutinin (HA) titers of IV, suggesting that the aggregation is also mediated by GPIIb/IIIa complex. The anti-GPIb monoclonal antibody (TM60) inhibited only the aggregation induced by low titers (HA: 5, 120 and 7, 680) of IV, but it could not inhibit the aggregation by high titers (HA: 10, 240) of IV. Removal of N-terminal 45 kDa fragment from GPIb by treatment with elastase resulted in the complete loss of ristocetin-induced aggregation but did not cause any decrease in IV-induced aggregation. The aggregation of chymotrypsin-treated platelets induced by IV or ristocetin gradually dereased with increasing of chymotrypsin. However the platelets pretreated with 10U/ml of chymotrypsin showed complete loss of ristocetin-induced aggregation but still had IV-induced aggregation, indicating that not only GPIb but also other components exhibited binding sites for IV. Treatment of the platelets with sialidase dose-dependently inhibited both high and low titers of IV-induced aggregation but had no effect on ristocetin-induced aggregation. IV-induced aggregation of sialidase-pretreated platelets was further decreased by TM60, while that of chymotrypsin-pretreated platelets was not inhibited.These results indicated that IV binds to not only sialic acid residue of GPIb with the highest affinity but also other sialic acid of the surface membrane with low affinity.

Effect of the Hirudin Carboxy-Terminal Peptide 54-65 on the Interaction of Thrombin with Platelets

The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed beta cleavage site, we have analyzed the consequences of alpha to beta-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The beta cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the beta cleavage site at Arg 73 on the thrombin B chain.

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).

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).

Comparison of Platelet Activation and Membrane Glycoprotein Ib and IIb‐IIIa Expression after Filtration through Three Different Leukocyte Removal Filters

We evaluated three filters used for leukocyte removal from platelet concentrates: Imugard IG 500, Pall PL100 and Sepacell PL-10A. Filter performance, platelet activation and expression of membrane glycoproteins Ib and IIb-IIIa were evaluated. Imugard, Pall and Sepacell showed median postfiltration in vitro platelet recoveries of 88, 84 and 80%, and total residual leukocyte counts of 16.1, 7.5 and 0.6 x 10(6)/pool of 8 platelet concentrates, respectively. Mean platelet volume was reduced after filtration with all filters. Postfiltration values of glycoproteins Ib and IIb-IIIa, and of activation markers GMP 140 and gp 53 were not significantly different from prefiltration values. Filtration through Imugard, Pall and Sepacell did not induce significant platelet activation or modifications of platelet membrane glycoproteins Ib and IIb-IIIa.