Expression of markers of platelet activation and the interpatient variation in response to abciximab

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.     

Molecular and genetic analysis of two patients with Bernard-Soulier syndrome--identification of new mutations in glycoprotein Ib alpha gene

We investigated two unrelated patients with Bernard-Soulier syndrome (BSS) by performing molecular and genetic analysis. A flow cytometric and immunoblotting analysis showed GP Ib alpha to be absent from the platelet membrane of both patients. Other glycoproteins that formed GP Ib/IX/V complex were present on the platelets, but in decreased amounts. Therefore, GP Ib alpha gene from both cases was sequenced after PCR amplification and subcloning. We identified a homozygous mutation of a dinucleotide deletion within the TGTG repeat at cDNA number 972 to 975 in GP Ib alpha gene from Case 1. In Case 2, compound heterozygosity was demonstrated in GP Ib alpha gene; an insertion of a single base (T) at cDNA number 1,418 in one allele, and a deletion of a single base (A) within the 7-adenine repeat at cDNA number 1,438 to 1,444 in another allele. The three new mutations in both patients appeared to cause a frameshift, which created a new termination codon shortly thereafter, and thus lead to a GP Ib alpha deficiency on the platelet membrane. Truncated mutant proteins could be detected in the plasma and platelets of Case 2, but not of Case 1. According to these findings, it is thus supposed that the properties and conformation of additional COOH-terminal peptides, which were supposedly synthesized as results of the mutations, may have an important role on the processing of mutant GP Ib alpha in megakaryocytes and platelets.     

Evaluation of ADCI against convulsant and locomotor stimulant effects of cocaine: comparison with the structural analogs dizocilpine and carbamazepine

Molecular genetic analysis has been performed on a patient with Bernard-Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Willebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ib alpha (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ib alpha and presence of small amounts of GP IX on the surface of the patient's platelets. In this study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the alpha-subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)-amplification of the genomic DNA coding for GP Ib alpha was performed. Nucleotide sequence analysis of the entire coding region of GP Ib alpha revealed a homozygous single base pair mutation T-->A, leading to a single amino acid substitution cysteine-->serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T-->A mutation, to analyse all available family members. PCR-ASRA (allele-specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to be a carrier of the mutant allele. The mutation identified in the family studied, responsible for the deficiency of the GP Ib/IX/V complex, suggests that the cysteine at amino acid position 209 may be involved in disulphide bonding.     

PAICA: a method for characterizing platelet-associated antibodies--its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA ("Platelet-Associated IgG Characterization Assay"), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient's own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA ("Monoclonal Antibody-specific Immobilization of Platelet Antigens"). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.     

A dinucleotide deletion results in defective membrane anchoring and circulating soluble glycoprotein Ib alpha in a novel form of Bernard-Soulier syndrome

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIb alpha, GPIb beta, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIb alpha was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIb alpha. This mutation (GPIb alpha deltaAT) causes a frame shift that alters the amino acid sequence of GPIb alpha within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIb alpha deltaAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIb alpha. Transient expression of recombinant GPIb alpha deltaAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIb alpha deltaAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GP beta-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIb alpha starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIb alpha polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIb alpha.     

Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide

In 1990 we reported that GP Ib-IX complexes accumulated within the surface-connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin-embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide.     

Hormonal therapy and genital tract cancer

Patient B.G. is a 29-yr-old female with a lifelong bleeding disorder characterized clinically by a highly increased bleeding time, menorrhagias, long-lasting bleeding after cuts and tooth extractions and large post-traumatic haematomas. Her coagulation tests were within normal range, platelet count was 140,000-160,000 per microliters, but platelet function was impaired as demonstrated by the absence of collagen-induced aggregation, although no abnormalities were detected in aggregation response to ADP and ristocetin. Morphologically her platelets were characterized by gigantic size-average profile area was about 2.5 times higher than that of control donors, and severe deficiency of alpha-granules-only 16% of their number in control donors. These features taken together indicated the diagnosis of grey platelet syndrome. As has been shown by quantitative immunoblotting, patient's platelets contained small amounts of alpha-granule membrane protein P-selectin-about 15% of that in control donors. The content of plasma membrane glycoproteins IIb-IIIa and Ib was not reduced, suggesting the specific deficiency of alpha-granule membrane protein. Thus, B.G. is the second patient described in the literature (see also Lages et al, J Clin Invest 1991: 87: 919-929) with combined deficiency of alpha-granules and P-selectin.     

Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.     

Comparative studies of glycoprotein Ib in heparin coated and nonheparin coated extracorporeal circulation circuits

Contact between blood and artificial materials has various effects on blood. Impairment of platelet function is an especially important and well known effect, but its precise mechanism is not clearly understood. The authors constructed a circulation model to investigate the effect of extracorporeal circulation on platelet membrane glycoproteins (GPs), especially GP Ib, and to compare the changes in GP Ib in heparin coated (group C) and nonheparin coated (group N) circuits. As determined by flow cytometry, GP Ib in both groups decreased on initiating circulation, but the decrease in group N was significantly larger than that in group C. There was no observed change in GP IIb/IIIa levels in either group. The extent of shear stress induced platelet aggregation significantly decreased during circulation in both groups. Decreases in the extent of shear stress induced platelet aggregation were significantly less with the use of heparin coated circuits. In addition, the amount of GP Ib in the high speed pellet decreased progressively during circulation in both groups. In contrast, the amount of GP Ib in the Triton insoluble (low speed) pellet increased dramatically during circulation. However, expression of GP Ib in the Triton soluble platelet fraction was low in both groups. From the results, it was concluded that the cause of the decrease in platelet function during extracorporeal circulation is attributable to the internalization of GP Ib from the platelet surface inside the platelet. It also can be said that a heparin coated circuit is one effective means of controlling this change.     

Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex

Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.     

Ultrastructural analysis of the distribution of the vitronectin receptor (alpha v beta 3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the alpha-granule membrane

The vitronectin receptor (VnR or alpha v beta 3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the beta 3 subunit in common: GP IIb-IIIa complexes (or alpha IIb beta 3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the alpha v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, alpha v beta 3 was mostly detected in internal membrane systems including those of alpha-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of alpha v beta 3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb-IIIa, although intracellular staining was present in EM and alpha-granules were again labelled.     

Prelabeled glycoprotein Ib/IX receptors are not cleared from exposed surfaces of thrombin-activated platelets

The present investigation has re-examined the hypothesis proposing that glycoprotein (GP)Ib/IX receptors for von Willebrand factor are rapidly cleared from exposed surfaces to internal membrane systems after activation of platelets by thrombin in suspension. Platelets were prelabeled with either a polyclonal antibody to GPIb alpha, antiglycocalicin (A-Gl), or a cocktail of two monoclonal antibodies, AP1 and 6D1, exposed to 0.1 or 0.2 U/ml thrombin for 5 or 10 minutes, fixed and stained with Staphylococcus protein A coupled to gold to detect A-Gl or goat anti-mouse IgG bound to gold particles to locate AP1 and 6D1 before or after preparation of frozen thin sections or embedding for plastic thin sections. The frequency and distribution of protein-A-gold markers for GPIb/IX on thrombin-activated platelets viewed in thin plastic sections did not differ from the density on resting platelets stained with A-Gl. Cryosections of A-Gl-prelabeled platelets labeled again on cryosections revealed GPIb present on linings of the open canalicular system of resting and activated platelets, but the density of gold in interior channels and frequency of gold particles on exterior surfaces were not altered by thrombin stimulation. Platelets prelabeled with the cocktail of 6D1 and AP1 and studied in cryosections also failed to reveal uptake of GPIb/IX receptors into the open canalicular system after activation by thrombin. The findings do not support the concept that thrombin causes clearance of GPIb/IX receptors from exterior surfaces to interior membranes of activated platelets.     

Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors

The blockade of platelet integrin glycoprotein (GP) IIb/IIIa is a promising new antiplatelet strategy. The binding of ligands or of the ligand-mimetic peptide RGD causes a conformational change of GP IIb/IIIa from the nonactivated to the activated state. Because several blocking agents/inhibitors are ligand-mimetics, the current study evaluates whether these agents have the intrinsic property to activate GP IIb/IIIa. Fibrinogen binding to GP IIb/IIIa on platelets or on CHO cells expressing recombinant GP IIb/IIIa was evaluated by flow cytometry or 125I-labeled fibrinogen. Incubation with the monoclonal antibody (MoAb) fragment c7E3 (abciximab) results in fibrinogen binding to GP IIb/IIIa and in the access of ligand-induced binding sites. At low concentrations (0.01 to 0.1 microgram/mL), this intrinsic activating property of c7E3 can result in platelet aggregation. The disintegrin flavorodin and the RGD analogue fradafiban also induce fibrinogen binding, whereas the blocking MoAbs 2G12 and P2 and the activation-specific MoAb PAC-1 do not. Aspirin and indomethacin cannot block c7E3-induced fibrinogen binding to GP IIb/IIIa, but can inhibit c7E3-induced platelet aggregation. Thus, we conclude that GP IIb/IIIa inhibitors can demonstrate an intrinsic activating property, which can result in fibrinogen binding to GP IIb/IIIa and consequently in platelet aggregation. Cyclooxygenase inhibitors can inhibit platelet aggregation caused by GP IIb/IIIa inhibitors. Further studies will have to evaluate the clinical relevance of the potential intrinsic activating property of GP IIb/IIIa inhibitors and define consequences for the future drug development and evaluation of these potent antiplatelet agents.     

Binding of collagen alpha1 chains to human platelets

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.     

Porcine platelet von Willebrand antigen II (vW AgII): inhibitory effect on collagen-induced aggregation and comparative distribution with human platelets

Von Willebrand factor (vWF) is synthesized by human endothelial cells and megakaryocytes as a large precursor, the pre-provWF, which is finally cleaved into the propolypeptide of vWF (pp-vWF) and the mature vWF. We have purified in parallel the pp-vWF and the GP IIb-IIIa from porcine platelets. The N-terminus comparative analysis of porcine pp-vWF with respect to other species revealed more than a 75% and 65% homology with the bovine and human pp-vWF, respectively. Purified pp-vWF inhibited collagen-induced platelet aggregation in porcine platelet rich plasma (PRP) and specifically binds to collagen. Polyclonal antibody pabBp19 against the purified protein was prepared and characterized by ELISA, Western-blot and immunocytochemistry. The distribution of pp-vWF in soluble and membrane fractions from pig platelets has been performed by immunolabeling detection. pabBp19 did not blot human platelet lysates but human pp-vWF was detectable using the antibody Frieda 013091. Cell distribution and quantification studies of human and porcine platelet pp-vWF showed that the protein exists in the soluble and membrane fractions and its pattern is similar in both species.     

Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa

Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.     

Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom of Crotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 microg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 microg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 +/- 0.1 x 10(-7) mol/L, and its binding site was estimated to be 58,632 +/- 3, 152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2 formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 microg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 microg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 microg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.