Formalin-fixed Platelets Research Articles

Functional Assessment of Platelet Dense Granule ATP Release.

This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 × 103/μL. Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.

Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254–A1461, which encompasses the C1272–C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Laboratory diagnosis and monitoring of desmopressin treatment of von Willebrand's disease by flow cytometry

von Willebrand's disease (VWD) is a heterogeneous bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The diagnosis of VWD requires several laboratory tests. The aim of our study was to validate a flow cytometric test for the diagnosis of VWD and for monitoring the effects of desmopressin therapy. Flow cytometric analysis of ristocetin-induced VWF binding to platelets was performed in platelet-rich plasma (PRP) samples from patients with VWD and from control subjects and in samples of formalin-fixed platelets in the presence of plasma from patients or controls. In 12 VWD patients the test was conducted before and 1 hour after desmopressin infusion. Results were compared with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA-100 and the skin bleeding time. Ristocetin-induced VWF binding to platelets, evaluated by both flow cytometry-based assays, was significantly reduced in patients with type1, 2A and 2M VWD as compared with that in healthy subjects. Patients with type 2B VWD showed reduced binding of VWF to formalin-fixed platelets, but increased binding to autologous platelets in PRP, similar to RIPA. VWF binding to platelets assessed by both flow cytometric assays correlated significantly with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA100 and bleeding time. VWF binding to platelets increased after desmopressin infusion. The measurement of ristocetin-induced binding of VWF to platelets by flow cytometry is a sensitive, simple and rapid test for the diagnosis of VWD and for the monitoring of the effects of desmopressin therapy. The flow cytometric assay performed with autologous platelets is useful in the identification of type 2B VWD patients.

Integrin αvβ3 Is Involved in the Stabilization of Ultra Long von Willebrand Factor Strings under Fluid Shear Stress on Human Endothelial Cells.

Von Willebrand factor (VWF) multimers attached to endothelial cells can provide a platform for thrombosis, especially when accompanied by ADAMTS13 deficiency. We characterized the structural features of ultralarge VWF (ULVWF) molecules acutely secreted from Weibel-Palade bodies and identified a receptor responsible for their binding to the surface of cultured human umbilical vein endothelial cells (HUVECs). Using fluorescence microscopy on live cells, VWF multimers formed extended strings within minutes after stimulation. String formation did not require exogenous platelets and occurred over a range of shear stress from 2.5 dyn/cm2 to 40 dyn/cm2. A subset of ULVWF strings spontaneously bound formalin-fixed platelets via platelet GPIb. Quick-freeze, deep-etch electron microscopy showed that ULVWF strings often merged to form bundles and networks. Each string was tethered to the endothelial membrane by a limited number of anchorage sites, many of them located on small membrane projections, suggesting a specific mode of interaction. Several independent approaches implicated integrin αvβ3 in anchoring ULVWF strings to the HUVEC surface. Either “RGDS” peptide or function blocking antibody (LM609) to integrin αvβ3 specifically and dose dependently inhibited ULVWF string formation 62 ± 0.7% and 53 ± 4%, respectively. Furthermore, integrin αv was seen decorating the extending ULVWF strings using a non-functional blocking antibody (LM142) in live-cell immunofluorescence. In addition, a lentiviral vector encoding shRNA against integrin αv resulted in approximately 70% reduction in cell surface αv expression by FACS analysis with antibody LM609. HUVEC infected with this lentivirus were significantly impaired in their ability to form ULVWF strings compared to cells infected with a control virus. In multiple experiments, shRNA knockdown of αv expression reduced ULVWF strings 75.3 ± 4.4% at 2.5dyn/cm2 and 81.6 ± 2% at 7.5dyn/cm2. These results indicated that ULVWF strings bind tightly to endothelial cells via very few anchorage sites and are relatively resistant to fluid shear stress. Although integrin αvβ3 does not appear to be required to stabilize ULVWF strings on mouse endothelium, these data suggest that integrin αvβ3 may participate in the stabilization of ULVWF strings on human endothelial cell surfaces.

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.

A Novel Tetrameric Venom Protein, Agglucetin from Agkistrodon acutus , Acts as a Glycoprotein Ib Agonist

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.

Hereditary defect of the platelet ADP receptor(s)

Four patients with a previously unrecognized congenital disorder of platelet function have recently been described. Their platelets aggregate very poorly to exogenous ADP. The abnormality is likely due to a severe defect of the platelet ADP receptor that is coupled to adenylate cyclase, as suggested by the following findings: 1) ADP does not normally lower cAMP levels of PGE1-treated platelets; 2) platelet shape change induced by ADP is normal; 3) the binding of \\[radiolabelled]ADP to formalin-fixed platelets or of the ADP analogue \\[radiolabelled]2-MeS-ADP to fresh platelets is severely defective. Since all patients that have been described were born from consanguineous parents, the condition seems to be inherited as an autosomal recessive trait. Platelets of an obligate heterozygote have intermediate binding sites for 2-M eS-ADP, undergo a normal primary wave of aggregation induced by exogenous ADP, but do not normally secrete the content of their granules when stimulated by release-inducing agonists. Studies of normal platelets treated with acetylsalycilic acid revealed that ADP potentiates platelet secretion directly, and that the full complement of its platelet receptors appears to be necessary for this function.

Quantitative defect of glycoprotein Ib in severe cirrhotic patients.

A decrease of platelet agglutination induced by ristocetin has been described in cirrhotic patients. In order to investigate the relationship of such phenomenon with a putative defect on platelet membrane glycoprotein Ib, we have studied the quantitative and functional status of Gp Ib in eleven severe alcoholic cirrhotic patients, and the ability of their formalin-fixed platelets to agglutinate in the presence of normal plasma plus ristocetin. Interestingly, we found a significant decrease of immunoreactive GP Ib molecules (9,978 +/- 1,534 vs. 17,064 +/- 404 molecules per platelet) and ristocetin-dependent binding of vWF (9,113 +/- 1,338 vs. 13,992 +/- 1,968 molecules per platelet) (P < 0.01) in comparison to the levels found in a control group of healthy subjects. Immunoblotting analysis of platelet lysates confirmed the reduction of GP Ib level in cirrhotic patients, but showed no modification on the precipitation pattern of this glycoprotein. The ristocetin-induced platelet agglutination (light transmission %) was also significantly lower in patients with cirrhosis (48 +/- 7.6 vs. 92 +/- 3.6, P < 0.01), and correlated with the binding of normal vWF (r = 0.863, P = 0.0013). Patients with bleeding times longer than 7 min and/or clinical history of bleeding episodes showed the lowest values of platelet agglutination and GP Ib level. In conclusion, our present results indicate that liver cirrhosis is associated with a relevant decrease of functional GP Ib molecules on the platelet surface. This reduction might be related to the prolongation of bleeding time and to the bleeding diathesis observed in cirrhotic patients.

Thrombospondin as an agglutinin of platelets

To study a hypothesis that thrombospondin (TSP) might function as an agglutinin in platelet aggregation, we designed two experiments. First, we prepared fibrinogen-coated agarose beads (fbg-beads) as a model of platelets, and subjected them to aggregometry using TSP as an inducer. TSP induced agglutination of fbg-beads in a dose-dependent manner. Calcium (Ca) and Magnesium (Mg) were necessary for the agglutination, and the aggregability was dependent on the concentration of Ca. These results confirmed the function of TSP as an agglutinin, suggesting some characteristics of the fbg-TSP interaction as well. Secondly, a variety of platelets were subjected to TSP-induced aggregation assay. Both gel-filtrated and washed-platelets were aggregated by TSP in a dose dependent manner and dissociated with EDTA. The same aggregation was observed in formalin-fixed platelets. Both Ca and Mg were required for the aggregation, and the maximum aggregation rate was dependent on the Ca concentration. Ca seemed to regulate the capacity as well as the affinity of the binding sites for TSP on platelets. Fibrinogen and some aminosugars inhibited the aggregation. These data suggest TSP may function as an agglutinin of platelets, and Ca may regulate the interaction between platelets and TSP. As one of the candidates for the receptor for TSP on platelet, fbg-GPIIb/IIIa was suggested because of the similarity between fbg-beads and platelets aggregation induced by TSP, and the Ca-dependency in both the GPIIb/IIIa induction and the TSP-induced platelet aggregation.

Anti‐fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb‐IIIa

The binding of fibrinogen to platelets requires the agonist activation of platelet membrane glycoprotein IIb/IIIa. We have now found an anti-fibrinogen polyclonal antibody (YCU-R3) that increases the fibrinogen affinity of GPIIb/IIIa-binding function (activation) and subsequent platelet aggregation. The addition of intact IgG, F(ab)2 fragments or Fab fragments induced platelet aggregation. The antibody-mediated fibrinogen binding was specific and saturable. This binding was inhibited by native fibrinogen, the RGDS peptide, the peptide of the C-terminus gamma chain of fibrinogen (gamma 397-411), and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The antibody-dependent fibrinogen binding was similar to that induced by ADP. Moreover, after pretreatment with the anti-fibrinogen antibody and fibrinogen, formalin-fixed platelets bound to fibrinogen saturably. These results suggest that this anti-fibrinogen antibody may function as partial agonist.

O-linked carbohydrate of recombinant von Willebrand factor influences ristocetin-induced binding to platelet glycoprotein 1b.

By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking O-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in O-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein 1b on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease.

Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis.

In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs). High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.

Characterization of Three Alboaggregins Purified from Trimeresurus albolabris Venom

Alboaggregins (AL-A, AL-B, AL-C) isolated from Trimeresurus albolabris snake venom represent a new family of proteins which bind to platelet glycoprotein Ib (GPIb). These alboaggregins were purified to homogeneity with ion exchange HPLC (Mono-Q column) and hydrophobic HPLC (TSK Phenyl-5PW column). On SDS-polyacrylamide gel electrophoresis, apparent molecular weights of AL-A, AL-B and AL-C were 52 kDa, 26 kDa, and 121 kDa respectively, under nonreducing conditions. Upon reduction, each alboaggregin showed two types of chains with apparent molecular weights in the range of 15-20 kDa. All three alboaggregins agglutinated formalin-fixed platelets. Agglutination activities and binding of labeled alboaggregins to GPIb were specifically inhibited by the monoclonal antibody AK2 which is directed against the 45 kDa N-terminal region on GPIb, but not by monoclonal antibodies against other epitopes on GPIb. 125I-alboaggregin binding to platelets was not altered by the presence of thrombin. Alboaggregins did not bind to GPIIb/IIIa. Alboaggregins were competitive inhibitors for 125I-bovine vWF binding to platelets. Mutual competition studies between AL-A, AL-B and AL-C for the binding of labeled bovine vWF and AL-B to platelets demonstrated that AL-B and AL-C had a significantly higher affinity than AL-A.

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.

Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation

Formalin-fixed platelets have been used to study the binding of adenine nucleotides in order to avoid the complications of nucleotide metabolism and to achieve steady-state binding. Sp-adenosine-5'-(1-thiotriphosphate) (Sp-ATP-alpha-S) binds to platelets at two sites (Kd1 3 nM; 31,000 sites/platelet; Kd2 200 nM; 300,000 sites/platelet) as compared with values for ADP under these conditions (Kd1 30 nM; 25,000 sites/platelet and Kd2 3 microM; 400,000 sites/platelet) (bound/total approximately 0.1). Competition binding experiments showed that both of the ATP-alpha-S sites were accessible to ADP and vice versa. [35S]ATP-alpha-S was photoaffinity cross-linked to unfixed platelets by direct irradiation with ultraviolet light. A single radiolabeled component (120 kDa) was identified and shown to be identical with the alpha subunit of GPIIb based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-GPIIb monoclonal antibodies, by isoelectric focusing (pI 4.5-5.5), by immunoaffinity adsorption using monoclonal anti-GPIIb/IIIa antibodies coupled to Sepharose, and by crossed immunoelectrophoresis. Amino-terminal sequencing of a tryptic fragment labeled with [35S]ATP-alpha-S identified an 18-kDa domain beginning at Tyr-198 in the primary sequence of GPIIb alpha. These studies demonstrate the presence of an adenine nucleotide-binding site on GPIIb alpha.

Dimeric ristocetin flocculates proteins, binds to platelets, and mediates von Willebrand factor-dependent agglutination of platelets

Ristocetin in aqueous solution dimerizes with an equilibrium dissociation constant of 5.0 x 10(-4) M, i.e. approximately 1.1 mg ml-1 (Waltho, J.P., and Williams, D. H. (1989) J. Am. Chem. Soc. 111, 2475-2480). At concentrations of about 1.0 mg ml-1 ristocetin flocculates many proteins, lyses platelets and, in the presence of von Willebrand factor, agglutinates both fresh and formalin-fixed platelets. Because ristocetin exists as both monomeric and dimeric species, we sought to determine which of these forms flocculates proteins and agglutinates platelets. We found that: 1) the initial rate of flocculation of certain proteins, 2) the initial rate of agglutination of formalin-fixed platelets, and 3) the binding of ristocetin to formalin-fixed platelets are higher order solely with respect to the concentration of ristocetin dimers. As to the operative mechanism, it appears that bifunctional dimers cross-link proteins that possess multiple copies of a common recognition site. Preliminary evidence indicates that a recognition site is a beta-turn of the form X-P-G-X'.

Reduction of platelet glycoprotein Ib in uraemia

Patients with uraemia have abnormal platelet function that may be partially corrected by haemodialysis, cryoprecipitate or 1-desamino-8-D-arginine vasopressin (DDAVP). We studied the platelet von Willebrand factor receptor, glycoprotein Ib (GPIb), and plasma von Willebrand factor (vWF) in uraemic patients undergoing chronic haemodialysis. Using the slope of agglutination of formalin-fixed platelets as an index of response to ristocetin (with a constant amount of normal plasma as a source of vWF), we found the response of platelets from uraemic patients, both before (2.7 +/- 1.5, n = 40) and after dialysis (1.2 +/- 1.2, n = 40) to be significantly less than that for normal controls (14.1 +/- 10.2, n = 20; P less than 0.001). In addition, the agglutination response of platelets obtained after dialysis was less than that of platelets obtained before dialysis (P less than 0.001). Immunoblotting demonstrated decreased or absent staining of glycocalicin, a subunit of GPIb, in platelet lysates from 25 patients. All platelet samples with reduced glycocalicin also had decreased responses to ristocetin. Tritium-labelled platelets from seven patients showed decreased labelling of a protein with an electrophoretic mobility equivalent to that of GPIb (140,000 daltons). In addition, platelets with the lowest levels of surface GPIb, as demonstrated by flow cytometry, also had decreased ristocetin agglutination and decreased staining on immunoblot. Levels of von Willebrand factor antigen and ristocetin cofactor in plasma from 10 patients were generally within the normal range, although postdialysis levels tended to be higher than pre-dialysis levels. The pre- and post-dialysis plasma vWF multimeric patterns were normal.

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

Enhanced botrocetin‐induced type iib von willebrand factor binding to platelet glycoprotein ib initiates hyperagglutination of normal platelets

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPIb induced by these two substances have been reported. We have recently shown that the GPIb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.