Bernard-Soulier Syndrome Platelets Research Articles

Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

AbstractIntroduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear.Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses.Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice.Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin. DisclosuresNo relevant conflicts of interest to declare.

Abstract 58: Delayed Atherosclerosis in a Mouse Model of Bernard-Soulier Syndrome is Independent of Glycoprotein Ibα Extracytoplasmic Domain Deficiency

The pathogenesis of atherosclerosis involves the interplay of blood, stromal and endothelial cells; platelet interactions with vascular endothelium and leukocytes promote atherosclerosis. Glycoprotein (GP) Iba is the ligand-binding subunit of the platelet GPIb-IX-V adhesion receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterized by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We found that Ldlr-/- mice reconstituted with GPIb a -/- as compared to wild type control developed delayed atherosclerosis associated with reduced platelet binding to blood myeloid cells and reduced accumulation of CD11b + and CD11c + myeloid cells in the aortas. Live imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in GPIb a -/- mice, which also showed decreased TNF in blood monocytes along with decreased TNF and IL12p35, but enhanced arginase1 in aortas. In contrast, Ldlr-/- mice reconstituted with chimeric IL-4R/ GPIb a-Tg bone marrow produce platelets expressing GPIb-IX-V without the GPIba extracytoplasmic domain but less abnormal with respect to size and count and showed atherosclerotic lesion sizes similar to control mice. In conclusion, reduced platelet interactions with myeloid cells and delayed onset of atherosclerosis are not caused by defective GPIba-ligand binding but may result from the low platelet count and, possibly, other functional defects of BSS platelets.

Phosphatidylserine exposure and other apoptotic‐like events in Bernard‐Soulier syndrome platelets

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation.

Platelets Having a W127X Mutation in GPIX Express Sufficient Residual Amounts of the GPIbα Chain to Support Ristocetin-Mediated Agglutination and Adhesion to VWF and Collagen Under Conditions of Flow.

A W127X mutation in platelet glycoprotein (GP)IX is the most common genetic defect in Japanese Bernard-Soulier Syndrome (BSS). Patients having this mutation are often misdiagnosed as having immune thrombocytopenia (ITP) because of residual expression of GPIbα on the platelet surface. The purpose of the present investigation was to determine whether the residual amount of GPIbα present on the surface of GPIXW127X BSS platelets might be sufficient to support adhesive functions of the GPIb complex. Flow cytometric analysis of platelets from our GPIXW127X BSS patient revealed 7–11% of the normal level of GPIbα (3,700 receptors/platelet), although the expression of GPIbα estimated by western blot analysis was only 1% of normal. Expression levels of other receptors such as GPIIb, GPVI, and α2 integrin were 3–4 times higher per platelet than that of normal controls–probably due to the increased size of the BSS platelets. To characterize the ability of GPIXW127X BSS platelets to bind VWF, we evaluated their agglutination in the presence of various concentrations of ristocetin, and found that ristocetin-induced agglutination was absent at 1.2 and 1.5 mg of ristocetin/ml, but present at 2.0 mg/ml. We then evaluated the ability of GPIXW127X BSS platelets to adhere under conditions of flow to immobilized VWF, type 1 collagen, and type III collagen. Whereas platelets derived from a Type I BSS patient having a TGTG deletion within the GPIbα gene in which GPIbα is completely absent (Thrombosis and Haemost 77:1055,1997) were unable to adhere to immobilized VWF under conditions of high shear, GPIXW127X BSS platelets bound well, demonstrating that the residual amount of GPIbα present GPIXW127X BSS platelets is sufficient to mediate adhesion to VWF. Finally, recent studies (J Thromb Haemost 5:797,2007) have shown that platelet adhesion to Type III collagen can be mediated by either interaction of GPIbα with VWF, or by interaction of the integrin α2β1 with collagen under high shear flow. In support of this notion, adhesion of GPIbαTGTG del BSS platelets to immobilized Type III collagen could be completely inhibited by addition of the anti-α2β1 blocking mAb, Gi9, while adhesion of GPIXW127X BSS platelets to Type III collagen was only marginally affected. Taken together, we conclude thatplatelets having a W127X mutation in GPIX express residual amounts of functional GPIbα, andboth GPIb-VWF and integrin α2β1-collagen interactions are physiologically important under conditions of high shear stress.Residual expression of GPIbα in GPIXW127X platelets, therefore, likely accounts for the relatively mild bleeding phenotype in this variant form of BSS.

Qualitative platelet disorders.

1. Larry D Brace, PhD[⇑][1] 1. is Associate Professor Emeritus, University of Illinois at Chicago, Chicago IL 1. Address for correspondence: Larry Brace PhD, Professor Emeritus, University of Illinois at Chicago, 11285 Plainfield Road, Indian Head Park IL 60525. (708) 246-7150. lbrace{at}uic.edu. Upon completion of this article, the reader will be able to: 1. recognize the clinical presentation of patients with dysfunctional platelets. 2. describe the defect in each of the following hereditary disorders: Glanzmann thrombasthenia and Bernard-Soulier syndrome (BSS). 3. distinguish among the following types of hereditary platelet disorders: membrane receptor abnormality, secretion disorder, and storage pool deficiency. 4. ror each of the inherited platelet disorders listed, name useful laboratory tests and recognize diagnostic results. 5. Discuss general mechanisms of action for antiplatelet drugs. 6. explain the effects of paraproteins on platelet function. 7. describe the effect of aspirin, clopidogrel, and αIIb/β3 inhibitors on platelet function. 8. discuss the mechanism of the platelet defects associated with myeloproliferative diseases, uremia, and liver disease. Mucocutaneous bleeding in a patient whose platelet count is normal suggests a disorder of platelet function. Congenital and acquired disorders may cause abnormalities in each phase of platelet function: adhesion, aggregation, and secretion. The qualitative disorders are detected and monitored using platelet aggregometry.1 Qualitative disorders are summarized in Table 1. Bernard-Soulier (giant platelet) syndrome Bernard-Soulier syndrome (BSS) usually manifests in infancy or childhood with mucocutaneous hemorrhage characteristic of defective platelet function: ecchymoses, epistaxis, and gingival bleeding. BSS is inherited as an autosomal recessive disorder in which the glycoprotein (GP) Ib/IX/V complex exhibits abnormal function. Heterozygotes with about 50% of normal levels of GP Ib, GP V, and GP IX have normal platelet function. Homozygotes have moderate to severe bleeding characterized by enlarged platelets, thrombocytopenia, and decreased platelet survival. Platelet counts range from 40,000/μL to near-normal.2 Platelets typically are five to eight μm in diameter although a few reach 20 μm. Viewed by electron microscopy, BSS platelets contain a larger number of cytoplasmic vacuoles and membrane complexes.3-5 Four glycoproteins are required to form the GP Ib/IX/V complex: GP Ibα, GP Ibβ, GP IX, and GP V. These are present in the ratio of 2:2:2:1. The gene for GP Ibα is located on chromosome 17, the gene for GP Ibβ is located on chromosome 22, and the genes for GP IX and GP V are on chromosome 3. For surface expression of the complex, it seems that synthesis of three proteins, GP Ibα, GP Ibβ, and GP IX,… ABBREVIATIONS: ADP = adenosine diphosphate; BSS = Bernard-Soulier syndrome; GP = glycoprotein; GSA = guanidinosuccinic oxide MPDs = myeloproliferative disorders; NO = nitric oxide; NSAIDs = non-steroidal anti-inflammatory drugs; TXA2 = thromboxane A2; VWD = von Willebrand disease; VWF = von Willebrand factor. Upon completion of this article, the reader will be able to: 1. recognize the clinical presentation of patients with dysfunctional platelets. 2. describe the defect in each of the following hereditary disorders: Glanzmann thrombasthenia and Bernard-Soulier syndrome (BSS). 3. distinguish among the following types of hereditary platelet disorders: membrane receptor abnormality, secretion disorder, and storage pool deficiency. 4. ror each of the inherited platelet disorders listed, name useful laboratory tests and recognize diagnostic results. 5. Discuss general mechanisms of action for antiplatelet drugs. 6. explain the effects of paraproteins on platelet function. 7. describe the effect of aspirin, clopidogrel, and αIIb/β3 inhibitors on platelet function. 8. discuss the mechanism of the platelet defects associated with myeloproliferative diseases, uremia, and liver disease. [1]: #corresp-1

Genetic characterization of patients with Bernard-Soulier syndrome and their relatives from Southern Iran

Bernard-Soulier syndrome (BSS) is a rare recessively inherited bleeding disorder caused by the deficiency of the platelet glycoprotein (Gp) complex Ib/IX/V that is the von Willebrand factor receptor on platelets. In patients suffering from BSS platelet adhesion is typically impaired, while platelet aggregation is normal; macrothrombocytopenia is a common feature. In this study three different families from Southern Iran were investigated. GpIb/IX/V platelet expression as detected by flow cytometry was less than 2% of normal in six cases and 12% in the remaining one. Platelet count was 35 000 platelets/microliter and iron deficiency anemia was common. All patients suffered from mucocutaneous bleeding at presentation and were born from consanguineous marriages. Genetic analysis demonstrated the presence of the same GpIX Phe55Ser missense mutation in two families and of a single base insertion (GP1BA C3221 ins), a never described mutation causing a frameshift in the GpIbα gene, in the third family. Among the family members studied several heterozygotes were identified. None of them, with one exception, had macrothrombocytopenia. In one family a slight reduction of GpIb/IX/V expression was observed.

Glycoproteins expression on platelet membrane in inherited macrothrombocytopenias

Introduction: Inherited giant platelet disorders (IGPD) are a heterogeneous group of rare diseases characterized by thrombocytopenia, large platelets and variable bleeding symptoms. Glycoprotein (GP) expression on platelet surface in these conditions is poorly characterized. We have investigated the expression of constitutively expressed platelet membrane GP and the response to TRAP activation in a group of patients with different forms of inherited macrothrombocytopenias. Materials and methods: Two patients diagnosed Epstein syndrome (ES), two with Bernard-Soulier syndrome (BSS) and eight with Mediterranean macrothrombocytopenia (MM) were studied using flow cytometry combined to monoclonal antibodies (MoAbs). Mean platelet volume was also measured. Results: In general, there was an increase in the binding of MoAbs directed against platelet membrane GPs in the patients studied (except, obviously, in BSS patients). The observed increase ranged between 0.9 and 3.36 times the value of the control for GPIbα, between 1.36 and 7.2 times for GPIIb–IIIa and 0.94 and 8.07 times for GPIV. However, when the observed value was divided by the measured platelet volume, the estimated density was similar to controls in the case of GPIbα but were increased for GPIIb–IIIa and GPIV. TRAP activation provoked significant increments in the number of copies of GPIIb–IIIa complexes present on platelet surface in controls (91%), MM patients (49%), but almost no changes in BSS platelets (6%). Conclusion: In the group of macrothrombocytopenia studied, an increase in the expression of platelet membrane GP was observed, although a wide variability exists even in patients with the same disorder.

Responses to Aggregating Agents after Cleavage of GPIb of Human Platelets by the O-Sialoglycoprotein Endoprotease from Pasteurella haemolytica— Potential Surrogates for Bernard-Soulier Platelets? ☆

Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave other glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectively cleaves the heavily O-glycosylated GPlb, but does not cleave N-linked glycoproteins or unglycosylated proteins. Isolated, [ 14C]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 μg/mL endoprotease for 60 minutes at 37°C. These platelets did not release [ 14C]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and release of [ 14C]serotonin by the endoprotease-pretreated platelets were inhibited in response to low concentrations of thrombin, SFLLRN (the PAR-1-activating peptide), collagen, and U46619 (a thromboxane A 2 mimetic); aggregates were smaller in size. The presence of fibrinogen overcame the inhibition of responses induced by SFLLRN, collagen, and U46619. With fibrinogen, primary ADP-induced aggregation was scarcely affected by pretreatment with the endoprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collagen, thromboxane A 2, fibrinogen (GPIIb/IIIa), and ADP appear to function normally on the endoprotease-pretreated platelets. Since only GPIb is cleaved by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet functions in this condition.

Defective thrombin-induced calcium changes and aggregation of Bernard-Soulier platelets are not associated with deficient moderate-affinity receptors.

Cloning of the moderate-affinity, serpentine thrombin receptor has helped clarify the mechanism of thrombin-induced platelet activation. Proteolytic cleavage by thrombin generates a new amino terminal that autostimulates the receptor, leading to activation of multiple signaling pathways and the platelet response. The function of other thrombin receptors, such as high-affinity glycoprotein Ib (GPIb), on platelets and their relationships to the moderate-affinity receptor remain unclear. The present study examined the role of the moderate-affinity thrombin receptor in Bernard-Soulier syndrome (BSS) platelets, which contain low amounts of GPIb. Platelets from four BSS subjects displayed normal aggregation profiles and cytosolic calcium changes in response to moderate or high concentrations of thrombin. In contrast, the BSS platelet aggregation response was delayed and calcium changes were absent in response to low thrombin concentrations. Platelets from an asymptomatic BSS heterozygote displayed an activation profile similar to those of control individuals. Specific activation of the moderate-affinity receptor by a synthetic peptide caused similar aggregation in platelets from all individuals. The synthetic peptide also elicited calcium responses in BSS platelets. Platelets from the BSS subjects and from an individual with the May-Hegglin anomaly showed increased expression of the moderate-affinity thrombin receptor by flow-cytometric analyses. These results suggest that BSS platelets possess high levels of a functional moderate-affinity thrombin receptor, probably due to large platelet size, and provide indirect evidence that a high-affinity thrombin receptor is associated with GPIb.

Glycoprotein llb-llla and Glycoprotein IV Expression on Bernard-Soulier Syndrome Platelets

Glycoprotein llb-llla and Glycoprotein IV Expression on Bernard-Soulier Syndrome Platelets

Inherited giant platelet disorders

Giant platelet disorders (GPD) refer to rare, usually inherited states characterized by abnormally large platelets, thrombocytopenia and bleeding tendency of variable severity. This review summarizes major clinical and laboratory features of three GPDs (Bernard-Soulier syndrome, May-Hegglin anomaly and gray platelet syndrome). Differential diagnosis between immunological thrombocytopenia and GPDs is important. Although rare, giant platelet disorders should be borne in mind, since bleeding tendency in some individuals may be severe and knowledge of bleeding diathesis is of importance before delivery or surgical procedures also in less symptomatic individuals.

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF-GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.

Characterization of Bernard-Soulier syndrome, Glanzmann's thrombasthenia using immunomagnetic cell separation technique

To characterize human platelet membrane glycoprotein (GP), we developed an immunomagnetic cell isolation method using monoclonal antibodies (MoAbs) that specifically bind to Ib and IIb-IIIa complexes of GP. When added to plateletrich plasma (PRP), anti-GPIb MoAb inhibited ristocetin-induced platelet aggregation, and anti-GPIIb-IIIa MoAb inhibited ADP- and collagen-induced platelet aggregation. Each MoAb was incubated with monosized polystyrene beads (4.5μm in diameter) coated with secondary antibody and then with normal PRP for 30 minutes. More than 90% of the platelets bound to the beads and were eliminated from PRP. However, the binding between the platelets and the beads coated with each MoAb was inhibited by pretreatment of PRP with the same type of MoAb.Platelets from patients with Bernard Soulier syndrome (BSS) and Glanzmann's thrombasthenia (GT) were added to the beads coated with each MoAb. BSS platelets with defective GPIb and GT platelets with defective GPIIb-IIIa did not bind to the beads coated with anti-GPIb MoAb and those coated with anti-GPIIb-IIIa MoAb, respectively.Our immunomagnetic isolation method using anti-GP MoAbs is very simple and useful for detecting abnormalities such as GP deficiency and especially diagnosing BSS and GT.

A Case of Bernard-Soulier Syndrome associated with von Willebrand Factor Antigen Abnormality

Bernard-Soulier syndrome (BSS), a function disorder of platelet with autosomal recessive inheritance, is characterized by relatively severe bleeding tendency, giant platelets accompanied by the deficiency of membrane glycoprotein Ib (GPIb) and thrombocytopenia. GPIb is thougt to play an important role in the process of thrombus formation through its adhesive activity to subendothelial tissue, interacting with von Willebrand factor (vWF). BSS platelets fail to aggregate in response to ristocetin despite normal content of factor VIII/vWF in plasma. In this paper, we report clinical and hematological data of a 5 year old homozygous female BSS case and her heterozygous parents. Laboratory examinations revealed moderately decreased amount of vWF antigen and molecular abnormality examined by the crossed immunoelectrophoresis and the SDS agarose gel electrophoresis in proband and her mother. The results of SDS polyacrylamide gel electrophoresis/PAS stain and immunomagnetic platelet separation with monoclonal antibody to GPIb/GPIIbIIIa showed a lack of GPIb in proband and complicated abnormalities of GPIIb/IIIa complex in her parents. Intracellular Ca2+ mobilization under the stimulation of thrombin showed markedly decreased levels in proband and about half amount of normal in her parents.

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the .alpha.-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.

Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets.

The interaction of the heparin-dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin-induced thrombocytopenia. Both normal platelets and Bernard-Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib. GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV.3) strongly inhibited the heparin-dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti-GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti-GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C-serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin-dependent antibody with platelets and heparin is mediated by a Fc-dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti-GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.

The mechanism of heparin‐induced platelet aggregation

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

4 Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare autosomal bleeding disorder characterized clinically by prolonged skin bleeding time, normal clot retraction and thrombocytopenia with large and morphologically abnormal platelets, and biochemically by the absence of platelet membrane glycoproteins (GP) Ib, V and IX. GP Ib and GP IX exist in the platelet membrane as a heterodimer complex which acts as the major receptor mediating platelet adhesion to blood vessel subendothelium. Studies with BSS platelets have proved particularly rewarding in the investigation of the GP Ib-IX complex as a multifunctional receptor protein. The transmembrane complex contains binding domains for von Willebrand factor, thrombin, fibrin and quinine/quinidine drug-dependent antibodies as well as an attachment site on the cytoplasmic side of the membrane for a platelet cytoskeleton. In addition, the internal segment of the beta-chain of GP Ib contains a cyclic AMP-dependent protein kinase-associated phosphorylation site which appears to regulate platelet reactivity. Limited proteolytic cleavage of the complex, in particular the GP Ib alpha-chain, has allowed immunological and functional characterization of three distinct domains; a 45 kDa segment at the N-terminal end of the alpha-chain of GP Ib, which contains binding sites for von Willebrand factor and thrombin, a 90 kDa highly glycosylated region of GP Ib alpha and a membrane-associated region consisting of the remnant of GP Ib alpha disulphide-linked to GP Ib beta and non-covalently-complexed with GP IX. This membrane-associated region contains the antigenic epitope(s) for quinine/quinidine drug-dependent antibodies. It is highly probable that the future study of platelets from patients with the Bernard-Soulier syndrome will further clarify the role of the GP Ib-IX complex in platelet physiology.

Flow cytometric analysis of platelet surface antigens.

A flow cytometric analysis of human platelet surface antigens was carried out using a panel of monoclonal antibody reagents. The reagents used were specific either for the GPIb or the GP IIb/IIIa complex, surface immunoglobulin, or von Willebrand factor (vWf). Indirect surface immunophenotypes were determined using an EPICS V flow cytometer and the monoclonal antibodies 6D1, 10E5, Plt-1, UR1663, anti-IgG, and anti-vWf. Platelets were obtained from normal individuals or patients with either Bernard-Soulier syndrome (BSS) or Glanzmann's thrombasthenia (GT). Normal platelets were positive for 10E5, 6D1, Plt-1, and UR1663 and showed negligible activity for anti-IgG and anti-vWf. Platelets from individuals with BSS showed a marked reduction in 6D1, while the platelets of a patient with GT showed a marked reduction in binding of 10E5, Plt-1, and UR1663. Differences between histograms for normal platelets and for platelets from individuals with BSS or GT were evaluated using the Kolmogorov-Smirnov test. Compared to normal platelets, the BSS and GT platelets contain at least 35-fold less of the GPIb and GP IIb/IIIa complex respectively. Flow cytometry is a useful and precise method for the study of normal and abnormal surface platelet phenotypes.

Human anti‐PlEl antibody recognizes epitopes associated with the alpha subunit of platelet glycoprotein Ib

Together, a platelet-reactive antibody in the serum of a polytransfused patient (proband) and a platelet-reactive antibody in the serum of a mother of an infant with neonatal thrombocytopenia have served to establish the diallelic, platelet-specific alloantigen system, PlE. We now provide evidence that the platelet-specific antibody in the serum of the proband, anti-PlE1, recognizes epitopes associated with the alpha subunit of glycoprotein (GP) Ib. By 51Cr release, platelets from two of three patients with the Bernard-Soulier syndrome (BSS) responded sub-normally to anti-PlE1, and the apparently normal response of platelets from the last BSS patient was attributable to anti-HLA-A2 antibodies in the proband serum. These results suggested that the PlE1 antigen is associated with the GPIb complex (glycoproteins Ib kX) known to be absent from BSS platelets. This possibility was confirmed by ELISA using the purified GPIb complex or glycocalicin, the N-terminal fragment of GPIb alpha produced by proteolysis with endogenous platelet calpain, as solid-phase antigen. Anti-PlE1 antibody bound specifically to both the GPIb complex and glycocalicin. 3H-labelled platelet membrane glycoproteins with apparent molecular weights of 130k, 25k, and 21k (under reduced conditions) corresponding to GPIb alpha, GPIb beta, and GPIX were immunoprecipitated by anti-PlE1 plasma. Finally, at a titre of 1:16, anti-PlEl completely inhibited ristocetin-induced platelet agglutination, a property of platelets mediated by GPIb.