Von Willebrand Factor Receptor Research Articles

Disappearance of glycoprotein Ib from the platelet surface in pericardial blood during cardiopulmonary bypass

Objectives: Several investigators have reported decreased expression of glycoprotein Ib on the platelet surface during coronary artery bypass grafting, but others could not confirm this finding. Because platelet glycoprotein Ib functions as an adhesion receptor for von Willebrand factor and other adhesive proteins, this decreased expression may explain excessive postoperative blood loss. In this study the expressions of glycoprotein Ib and other platelet activation markers were studied in the systemic and pericardial blood of seven patients undergoing coronary artery bypass grafting. Pericardial blood was recently shown to have high activation levels of fibrinolytic and coagulation pathways; we hypothesized that this local blood activation might be paralleled by extensive platelet activation and associated disappearance of glycoprotein Ib. Methods: Expression of platelet surface antigens was determined by whole-blood double-label flow cytometry. Results: Glycoprotein Ib expression in systemic blood decreased 10% ( p = 0.03) from preoperative levels at the start of cardiopulmonary bypass and 30% ( p = 0.04) before release of the aortic crossclamp. Expression in pericardial blood at these times decreased by 50% and 51%, respectively ( p = 0.003, p = 0.009). No changes were observed in the expression of the platelet activation antigens CD62P (P-selectin, indicating platelet α-granular release) and CD63 (indicating lysosomal release) or in binding of monoclonal antibody PAC-1 (detecting the fibrinogen-binding receptor conformation of the glycoprotein IIb-IIIa complex). Conclusion: Glycoprotein Ib disappeared from the platelet surface during bypass grafting, most notably in pericardial blood. No increased expression of CD62P, CD63, or PAC-1 was found, indicating the absence of general platelet activation. (J Thorac Cardiovasc Surg 1998;115:1160-5)

Platelet glycoprotein receptor IIIa polymorphism P1A2 and ischemic stroke risk: the Stroke Prevention in Young Women Study.

Platelet glycoprotein IIb/IIa (GpIIb-IIIa), a membrane receptor for fibrinogen and von Willebrand factor, has been implicated in the pathogenesis of acute coronary syndromes but has not been previously investigated in relation to stroke in young adults. We used a population-based case-control design to examine the association of the GpIIIa polymorphism P1A2 with stroke in young women. Subjects were 65 cerebral infarction cases (18 patients with and 47 without an identified probable etiology) 15 to 44 years of age from the Baltimore-Washington region and 122 controls frequency matched by age from the same geographic area. A face-to-face interview for vascular disease risk factors and a blood sample for the P1A2 allele and serum cholesterol were obtained from each participant. Logistic regression was used to estimate the odds ratio for one or more P1A2 alleles after adjustment for other risk factors. Among cases and controls, the prevalence rates of one or more P1A2 alleles were 21% and 22% among blacks and 36% and 28% among whites, respectively. This genotype was significantly associated with hypertension only in black control subjects but otherwise not with any of the established vascular risk factors. The adjusted odds ratio for cerebral infarction of one or more P1A2 alleles was 1.1 (confidence interval [CI], 0.6 to 2.3) overall, 0.5 (CI, 0.1 to 7.1) among blacks, and 1.4 (CI, 0.5 to 3.7) among whites. For the cases with an identified probable etiology, the corresponding odds ratios were 3.0 (CI, 0.9 to 10.4) overall, 0.7 (CI, 0.1 to 7.1) among blacks, and 12.8 (CI, 1.2 to 135.0) among whites. No association was found between the P1A2 polymorphism of GpIIIa and young women with stroke. However, subgroup analyses showed that the P1A2 polymorphism of GpIIIa appeared to be associated with stroke risk among white women, particularly those with a clinically identified probable etiology for their stroke. Further work with an emphasis on stroke subtypes and with multiracial populations is warranted.

Association of the Platelet Glycoprotein IIIa PIA1/A2 Gene Polymorphism to Coronary Artery Disease but not to Nonfatal Myocardial Infarction in Low Risk Patients

The platelet membrane glycoprotein IIb/IIIa functions as a receptor for fibrinogen and von Willebrand factor during platelet aggregation. In a small case-control study, evidence has been presented that the PlA2 allele of the platelet glycoprotein GPIIIa PlA/A2 gene polymorphism might be an independent risk factor for acute myocardial infarction (MI). We explored the association of the PlA1A2 to the severity of coronary artery disease (CAD), as assessed angiographically in 2252 male individuals, and to myocardial infarction (MI). The severity of coronary heart disease (CHD) was also estimated by calculating a CHD score according to Gensini. The PlA genotype was determined by allele specific restriction digestion. Relation of the PlA2 allele to CAD: In the total population, the frequency of the PlA2 allele was not associated to the presence or to the extent of CAD. Also the CHD scores of PlA1/PlA2 genotypes were essentially the same. However, after exclusion of individuals with high BMI (> or =26.9 kg/m2) and/or low apoAI (< 1.43 g/l) PlA2PlA2 carriers had clearly higher CHD scores than PlA1PlA1 genotypes: PlA1PlA2 heterozygotes had intermediate values (p <0.05). After division of the study population into one group of individuals without any angiographic signs of CAD (CHD score = 0) and into another group of patients with severe CAD (CHD score (> or = 120), a strong association of the PlA2 allele with severe CAD was also found in the same low risk groups: e.g. exclusion of persons with high BMI and low apoAI resulted in an Odds ratio of 5.37 (1.46-19.7) (p <0.02). Relation of the PlA2 allele to MI: No association was found between PlA1/PlA2 genotypes and risk of MI neither in the total population nor in low risk subgroups. Whereas no difference in the distribution of allele and genotype frequencies between controls and survivors of MI could be detected, the PlA2 allele is associated with CHD in low risk patients.

Coronary Artery Disease and Polymorphisms in a Receptor Mediating Shear Stress–Dependent Platelet Activation

Platelets play pivotal roles in coronary thrombosis, and antiplatelet therapies are widely used for coronary artery disease (CAD). However, the effects of genetic variation in platelets on CAD are poorly understood. We have assessed the association between CAD and polymorphisms in a platelet receptor for von Willebrand factor, the glycoprotein (GP) Ib/IX complex, which mediates shear stress-dependent platelet activation. Genotypes of the alpha-chain of the receptor (GP Ib alpha, 145Thr/Met) were determined in 91 patients with myocardial infarction (MI) or angina pectoris whose lesions were confirmed by coronary angiography as well as in 105 individuals from the general population with no history of angina or other heart diseases and normal resting ECGs. There was no homozygote for Met/Met in either the control or patient groups. The prevalence of the Thr/Met genotype (T/M) in all patients was not significantly different from that in the control group. However, the frequency of T/M was significantly higher in patients aged < or = 60 years (31.8%) than in control subjects aged < or = 60 years (16.0%; P<.05, odds ratio=2.5). An association was also demonstrated between CAD and the other polymorphism of GP Ib alpha, a variable number of tandem repeats of a 13-amino acid sequence, which is known to be linked to the 145Thr/Met polymorphism. There was an association between the frequency of the T/M genotype and the angiographic severity of CAD: 11.1% for Gensini score < 40 versus 50.0% for Gensini score > or = 40 (P=.0015). There was no difference in the distribution of GP Ib alpha genotypes between patients with MI and those with angina pectoris. This study suggests that the presence of the Met allele in GP Ib alpha is a risk factor for the prevalence and severity of CAD in individuals aged < or = 60 years. The results need to be confirmed in a large-scale study of incident case subjects and matching control subjects.

A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα 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: GPIbα, GPIbβ, 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 GPIbα 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 GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα 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 GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-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 GPIbα 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 GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.

A Dinucleotide Deletion Results in Defective Membrane Anchoring and Circulating Soluble Glycoprotein Ibα in a Novel Form of Bernard-Soulier Syndrome

AbstractThe platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, 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 GPIbα 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 GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα 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 GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-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 GPIbα 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 GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.

Flow cytometric analysis of platelet activation throughout normal gestation

To measure platelet activation in normal pregnancy, before and after stimulation with agonists, with a whole blood flow cytometric technique. In a cross-sectional study, 5 mL of whole blood was collected from healthy volunteers (nine in the first trimester, ten in the second trimester, 35 in the third trimester, and 32 nonpregnant controls). Platelets were treated with an agonist (thrombin or U-46619, a thromboxane A2 analogue) or buffer and were exposed to saturating concentrations of monoclonal antibodies directed against platelet membrane glycoproteins (GPs): 7E3 (fibrinogen receptor GPIIb/IIIa), S12 (alpha granule marker P-selectin), and 6D1 (von Willebrand factor receptor GPIb). Mean fluorescence intensity was determined for 5000 platelets per sample by using a flow cytometer. In the absence of agonist, no significant difference between groups was found in antibody binding. At no stage of pregnancy were circulating activated platelets detected. Platelets from third-trimester subjects bound significantly less 7E3 than platelets of controls or of first- or second-trimester subjects after stimulation with high-dose thrombin (P < .05 for all comparisons). Down-regulation of 6D1 on platelets after stimulation with high-dose U-46619 was significantly greater in third-trimester gravidas than in controls or first-trimester subjects (P < .05). Pregnancy does not increase the percentage of activated platelets in the circulation. Platelet reactivity is altered in the third trimester, as evidenced by decreased antibody binding to fibrinogen receptor epitope and enhanced down-regulation of a von Willebrand factor receptor epitope.

CHARACTERIZATION OF THE GENE ENCODING MOUSE PLATELET GLYCOPROTEIN Ibβ

Platelet glycoprotein (GP) Ib/IX/V complex is a major receptor for von Willebrand factor (vWF), which mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of GPIbα, GPIbβ, GPIX, and GPV. All subunits for the human receptor have been cloned and characterized. However, the function of GPIbβ is still elusive. To obtain further information of GPIbβ, we have determined the genomic sequence of mouse GPIbβ (1466 bp). The deduced amino acid sequence (206aa) was 88% identical to the human GPIbβ protein. All cysteine residues, putative N-linked glycosylation site (Asn41), and putative phosphorylation site (Ser166) were conserved. The promoter region contained putative GATA and ets binding motif implicated in megakaryocytic expression. Mouse GPIbβ also contained a leucine-rich glycoprotein (LRG) sequence of 24 amino acids same as human GPIbβ. © 1997 Elsevier Science Ltd

Polymorphisms of the human platelet antigens HPA-1, HPA-2, HPA-3, and HPA-5 on the platelet receptors for fibrinogen (GPIIb/IIIa), von Willebrand factor (GPIb/IX), and collagen (GPIa/IIa) are not correlated with an increased risk for stroke.

A recent study has described a high incidence of the human platelet antigen (HPA)-1b alloantigen in patients with myocardial infarction. We investigated the distribution of gene polymorphisms of platelet glycoproteins (GPs) in patients with cerebrovascular disease (CVD) and stroke. The polymorphic systems we have studied are HPA-1 and HPA-3 on the fibrinogen receptor (GPIIb/IIIa), HPA-2 on the von Willebrand factor receptor (GPIb/IX), and HPA-5 on one of the platelet collagen receptors (GPIa/IIa). DNA was isolated from peripheral blood collected from 218 consecutive stroke patients, 165 neurological inpatients without signs of CVD, and 321 healthy blood donors. The genotypes of HPA-1, HPA-2, HPA-3, and HPA-5 were determined by sequence specific primer polymerase chain reactions. The calculated allele frequencies were as follows: for CVD patients, HPA-1a/b 0.81/0.19, HPA-2a/b 0.91/0.09, HPA-3a/b 0.61/0.39, and HPA-5a/b 0.92/0.08; for inpatient HPA-1a/b 0.83/0.17, HPA-2a/b 0.91/0.09, HPA-3a/b 0.62/0.3 and HPA-5a/b 0.93/0.07; and for blood donors, HPA-1a 0.85/0.15, HPA-2a/b 0.94/0.06, HPA-3a/b 0.60/0.40, and HPA 5a/b 0.92/0.08. There were no statistically significant difference for the analyzed HPA polymorphism frequencies either between the CVD patients and the non-CVD inpatients or the CVD patients and blood donors. However, the HPA-1b genotype was slightly more frequent in patients (CVD and non-CVD) than in the healthy blood donors. Our results indicate that the HPA-1, HPA-2, HPA-3, and HPA-5 polymorphisms are not associated with an increased risk for stroke.

Cytokine Treatment of Endothelial Cells Increases Glycoprotein Ibα-Dependent Adhesion to von Willebrand Factor

AbstractEndothelial cells (EC) possess at least two membrane receptors for von Willebrand factor (vWF ), the vitronectin receptor (VNR, αvβ3 ), which recognizes an Arg-Gly-Asp (RGD) sequence in the C-terminus of vWF, and glycoprotein Ibα (GP Ibα), which interacts with a region in the N-terminal A1 domain of vWF. In the absence of added cytokines, EC attachment to a vWF substratum is mediated largely through the αvβ3 , with a smaller contribution by GP Ibα. In the present study, we have examined the effect of cytokines on the receptor specificity of EC attachment to wild-type vWF (WT-vWF ) and to vWF, which had been mutated in the C-terminal RGDS sequence (RADS-vWF ). Exposure of human umbilical vein EC (HUVEC) to tumor necrosis factor-α (TNF-α) or to TNF-α in combination with interferon-γ (IFN-γ), but not to interleukin-1β (IL-1), increased attachment to RADS-vWF by about twofold. The TNF-α–induced increase in EC attachment was accompanied by an increase in cell surface GP Ibα expression; GP Ibα surface expression was not increased by IL-1. Attachment of untreated HUVEC to WT-vWF could be inhibited 60% to 70% by a monoclonal antibody (MoAb) (LM609) to the VNR and 30% to 40% by the A1 fragment of vWF (containing the GP Ibα binding domain). The pattern of inhibition of attachment to WT-vWF was largely unchanged after TNF-α treatment of HUVEC. In contrast, the attachment to WT-vWF of HUVEC, treated with TNF-α +IFN-γ was completely inhibited by vWF-A1 and inhibited only 35% by the anti-VNR antibody LM609. Two MoAbs to GP Ibα produced similar, but incomplete, inhibition. Pretreatment of HUVEC with the combination of TNF-α +IFN-γ produced a dramatic decrease in VNR expression, confirming previous findings of Defilippi et al. These results suggest that in the presence of the inflammatory cytokines TNF-α +IFN-γ, the endothelial GP Ib complex is a major determinant of HUVEC adhesion to surface-bound vWF.

Gene Cloning of Rat and Mouse Platelet Glycoprotein V: Identification of Megakaryocyte-Specific Promoters and Demonstration of Functional Thrombin Cleavage

AbstractPlatelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V–IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (≈1,700 nucleotides) entirely contained in one exon and a single intron (≈900 nucleotides) in the 5′ untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8–amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2 -terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2 -terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2 -terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

Alternative expression of platelet glycoprotein Ib(beta) mRNA from an adjacent 5' gene with an imperfect polyadenylation signal sequence.

Glycoprotein (GP) Ib is a major component of the platelet membrane receptor for von Willebrand factor, designated the GP Ib-IX-V complex. GP Ib is composed of two subunits (GP Ib(alpha) and GP Ib(beta)) each synthesized from separate genes. The 206 amino acid precursor of GP Ib(beta) is synthesized from a 1.0-kb mRNA expressed by megakaryocytes and was originally characterized from cDNA clones of human erythroleukemic (HEL) cell mRNA, a cell line exhibiting megakaryocytic-like properties. The cell line CHRF-288-11 also exhibits megakaryocytic-like properties, but synthesizes two related GP Ib(beta) mRNA species of 3.5 and 1.0 kb. We performed cDNA cloning experiments to identify the origin of the 3.5-kb transcript and determine its relationship to the 1.0-kb GP Ib(beta) mRNA found in megakaryocytes, platelets, and HEL cells. Our cloning experiments demonstrate that the longer transcript results from a nonconsensus polyadenylation recognition sequence, 5'AACAAT3', within a separate gene located upstream to the platelet GP Ib(beta) gene. In the absence of normal polyadenylation the more 5' gene uses the polyadenylation site within its 3' neighbor, the platelet GP Ib(beta) gene. This newly identified 5' gene contains an open reading frame encoding 369 amino acids with a high degree of sequence similarity to an expanding family of GTP-binding proteins.

Influence of cytochalasin B (CB) on GP Ib distribution after thrombin or TRAP and before surface activation

The receptor for von Willebrand factor (vWF) on human platelets, glycoprotein (GP) Ib/IX, has been shown in our studies to be an immobile complex when stimulated in suspension or on surfaces. Recent investigations have revealed that GP Ib/IX remains immobile on platelets activated in suspension followed by exposure to formvar surfaces that cause the cells to spread. However, since channels of the open canalicular system (OCS) are evaginated back on to the exposed surface during spreading, it was suggested that our study missed the clearance of GP Ib/IX from the exposed surface to internal membranes. The present study has added cytochalasin B after exposure of platelets to thrombin or TRAP in suspension in order to prevent spreading and movement of GP Ib/IX during subsequent exposure to surface activation on formvar grids. Results indicate that GP Ib/IX receptors remain randomly dispersed from edge to edge on platelets activated by thrombin or TRAP in suspension 10 minutes before treatment with CB followed by surface activation. Statistical analysis of the frequency of immunogold particles binding to monoclonal antibodies attached to GP Ib/IX revealed no significant reduction in frequency, translocation from cell edges or concentration of GP Ib/IX receptors in or around channels of the OCS. Results support the concept that GP Ib/IX is not cleared from exposed surfaces to the OCS of platelets activated by thrombin or TRAP and surface activation.

Effect of a New Monoclonal Anti-Glycoprotein IX Antibody, KMP-9, on High Shear-Induced Platelet Aggregation

Human platelet glycoprotein Ib/IX complex acts as a receptor for von Willebrand factor. It is widely accepted that glycoprotein Ib is the essential receptor component, but the role of glycoprotein IX is still unclear. We produced a new monoclonal anti-glycoprotein IX antibody (KMP-9) by the hybridoma technique using platelets from a patient with Glanzmann's thrombasthenia. The epitope of KMP-9 was localized to the C-terminal 8 kD fragment of glycoprotein IX using ELISA analysis of polyethylene-pin-synthesized peptides, as well as Western blot analysis of platelets after digestion with N-glycosidase and Staphylococcus aureus V8 protease. KMP-9 partially inhibited high shear stress-induced platelet aggregation, but had no effect on aggregation induced by ristocetin or low shear stress. Its inhibitory effect on high shear stress-induced aggregation was weaker than that of anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa monoclonal antibodies. A 21-mer synthetic peptide (glycoprotein IX L110-G130) inhibited the binding of KMP-9 to platelets. It also competively inhibited the suppression of high shear stress-induced platelet aggregation by KMP-9, but had no direct effect on this aggregation. KMP-9 may be useful to clarify the physiological role of GPIX.

AGGREGATED-DISAGGREGATED, REFRACTORY PLATELETS RETAIN SENSITIVITY TO RISTOCETIN

The present investigation has used an aggregation-disaggregation-reaggregation model to determine if exposure to potent aggregating agents causing a refractory state in deaggregated platelets results in down-regulation and clearance of GPIb/IX, the receptor for von Willebrand factor. Thrombin, the thrombin receptor activating peptide (TRAP) and the thromboxane A 2 mimic, U46619, caused irreversible aggregation and secretion when stirred on an aggregometer with platelet-rich plasma (PRP). Addition of prostaglandin E 1 (PGE 1) after the plateau of maximum aggregation was reached caused rapid dissociation of platelet aggregates. Dissociated platelets were refractory to a second exposure to the primary stimulus or to other aggregating agents. Exposure of the refractory cells to epinephrine before the primary agent restored sensitivity resulting in a second wave of irreversible aggregation. Deaggregated, refractory platelets, however, retained their sensitivity to ristocetin. Amounts of the antibiotic causing agglutination of resting PRP also caused agglutination of disaggregated, refractory platelets. Addition of epinephrine to samples of refractory platelets less sensitive to low concentrations of the antibiotic restored their capacity to undergo irreversible ristocetin-induced agglutination. Analysis of the frequency of gold particles associated with monoclonal antibodies directed against GPIb/IX on control platelets and disaggregated, refractory platelets showed no significant difference in the number of receptors. The findings indicate that significant numbers of GPIb/IX receptors remain on platelet surfaces following exposure to potent aggregating agents. Copyright © 1996 Elsevier Science Ltd

A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis: Weiss EJ, Bray PF, Tayback M, et al. N Engl J Med 334:1090–1094, 1996

Background. Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggrega- tion. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, Pl A2 , of the gene encoding glycoprotein IIIa in kindreds with a high preva- lence of premature myocardial infarction. Methods. To investigate the relation between the Pl A2 polymorphism and acute coronary syndromes, we con- ducted a case-control study of 71 case patients with my- ocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the Pl A genotype: reverse dot blot hybridiza- tion and allele-specific restriction digestion. Results. The prevalence of Pl A2 was 2.1 times higher

Arteriovenous Fistula Obstruction and Expression of Platelet Receptors for Von Willebrand Factor and for Fibrinogen (Glycoproteins GPIb and GPIIb/llla) in Hemodialysis Patients

Platelet surface receptors for von Willebrand factor and for fibrinogen (glycoproteins GPIb and GPIIb/IIIa) were studied with monoclonal antibodies CD42 and CD41 and cytofluorometry in 31 healthy subjects, 10 hemodialysis patients with no A-V fistula obstruction (patent original fistula), 10 hemodialysis patients with frequent A-V fistula obstruction (more than twice), 12 patients with mild chronic renal failure (creatinine 1.75 +/- 0.40 mg/100 ml), 11 patients with advanced chronic renal failure (creatinine 5.62 +/- 1.22 mg/100 ml), and 10 patients with end-stage renal disease (ESRD) treated with peritoneal dialysis. There was a significant increase of platelet surface glycoproteins GPIb and GPIIb/IIIa in the population of hemodialysis patients with frequent A-V fistula obstruction. The expression of these platelet receptors might be related to the prothrombotic tendency of a group of patients with ESRD, who suffer more occlusive and thrombotic events of the A-V fistula. This group of patients may also have a higher frequency of systemic thrombotic and atherosclerotic complications.

Brief communications Aprotinin and aortic cannula thrombosis

Aprotinin has been advocated to reduce perioperative blood loss in patients undergoing cardiopulmonary bypass (CPB).I, 2 Its effect on the coagulation cascade has been investigated by many researchers and its clinical effectiveness has been documented. 3'4 However, the potential adverse sequelae of potent antifibrinolytic therapy has received less attention. Thrombus formation has been reported with pulmonary artery catheters after aprotinin administration. 5 Thrombus formation in the aortic outflow cannula, reported herein, represents a unique complication of antifibrinolytic therapy and merits attention. Case report. A 64-year-old woman weighing 78 kg, with end-stage congestive cardiomyopathy, underwent cardiac transplantation. Two million kallikrein inactivator units (FdU) of aprotinin was bolused intravenously before the initial skin incision, followed by a continuous infusion at 500,000 KIU/hr. Additionally, another 2 million KIU of aprotinin was added to the extracorporeal circuit as a priming dose before institution of CPB. Before CPB, heparin (4 mg/kg) was administered and an activated clotting time (ACT) of 600 seconds obtained. (ACT was measured in celite test tubes in an automated device [Hemochron, International Technidyne Corp., Edison, N.J.]. ACTs during CPB ranged between 600 and 963 seconds.) After implantation of an 18-year-old donor heart, the patient had cardiac graft dysfunction resulting in cardiogenic shock and hypotension. Mechanical support with intraaortic balloon counterpulsation and aggressive inotropic support were instituted. The patient was weaned from CPB and protamine was administered. After decannulation and observation in the operating room for 4 hours, the patient's cardiac hcmodynamics worsened. She was therefore reheparinized (4 mg/kg) and, with the aid of CPB, ABIOMED BVS 5000 (ABIOMED Cardiovascular, Inc., Danvers, Mass.) biventricular assist devices (BVADs) were inserted. A new extracorporeal circuit and new cannulas were used. Aprotinin was not rebolused or added to the prime of the new CPB circuit,

Effects of nitric oxide/EDRF on platelet surface glycoproteins.

We examined the effects of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) on platelet surface glycoproteins (GP). As determined by flow cytometry, in both a washed platelet system and platelet-rich plasma, the EDRF congener (S-nitroso-N-acetylcysteine) markedly inhibited both the thrombin-induced and the (stable thromboxane A2 analogue) U-46619-induced upregulation of P-selectin (alpha-granule protein), CD63 (lysosomal protein), and the GPIIb-IIIa complex (fibrinogen receptor) but minimally inhibited downregulation of the GPIb-IX complex (von Willebrand factor receptor). The inhibitory effects of EDRF were markedly reduced in whole blood or by the addition of washed erythrocytes. Platelets in whole blood were still responsive to guanosine 3',5'-cyclic monophosphate (cGMP), as shown by complete inhibition of P-selectin upregulation by the stable analogue N6,2'-O dibutyryl cGMP. These data suggests that 1) cGMP negatively regulates the platelet surface expression of P-selectin, CD63, and the GPIIb-IIIa complex but not the platelet surface expression of the GPIb-IX complex and 2) hemoglobin within erythrocytes inhibits the effects of EDRF/NO on platelet surface glycoproteins.

A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis.

Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction. To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion. The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4). We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.