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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Cadroy, Y. Articles by Sie, P. Search for Related Content PubMed PubMed Citation Articles by Cadroy, Y. Articles by Sie, P. Related Collections Hemostasis, Thrombosis, and Vascular Biology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 November 2001, Vol. 98, No. 10, pp. 3159-3161 BRIEF REPORT Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men Yves Cadroy, Kjell S. Sakariassen, Jean-Paul Charlet, Claire Thalamas, Bernard Boneu, and Pierre Sie From the Laboratoire de Recherche sur l'Hémostase et la Thrombose, Pavillon Lefèbvre, and Centre d'Investigation Clinique, CHU Purpan, Cedex, France; Department of Biology, Division of General Physiology, University of Oslo, Oslo, Norway; and Département d'Informatique, Hôpital La Grave, Toulouse, France.     Abstract Top Abstract Introduction Study design Results and discussion References This study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIb, the 5T/C polymorphism in the Kozak sequence of GPIb, the 807C/T polymorphism of GPIa, and the PlA1/PlA2 polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 s1. At 2600 s1 platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 s1 thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PlA1/PlA2 polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIb and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate. (Blood. 2001;98:3159-3161) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Arterial thrombosis is a process that involves platelet adhesion and aggregation. These events are mediated through the interactions of platelet membrane glycoproteins (GPs) with adhesive substrates. Platelet adhesion to collagen is one of the first steps of thrombus formation. von Willebrand factor mediates an initial tethering of platelets to collagen via GPIb. Platelets translocate along the collagen surface until movement is arrested by firm engagement of the platelet membrane collagen receptor GPIa-IIa (integrin 21). As a consequence of platelet activation and inside-out signaling, GPIIb-IIIa (integrin IIb3) acquires the ability to bind fluid phase fibrinogen or von Willebrand factor and thereby trigger platelet aggregation. The 3 major platelet membrane-adhesive receptors GPIb, GPIa-IIa, and GPIIb-IIIa present genetic polymorphisms. A number of clinical studies have reported conflicting data on the association of these polymorphisms and arterial thrombosis.1,2 However, these studies were often too small and heterogeneous with regard to the type of patients involved, the pathology, and the treatment. Information directly regarding the effect of these genetic changes on experimental thrombosis is lacking. In addition, no study has compared the respective role of these important genetic factors thought to be associated with an increased tendency to thrombosis. The aim of the present study was to assess the role of 4 polymorphisms of these 3 platelet receptors that potentially are linked to an increased thrombotic tendency. We used an experimental model of arterial thrombus formation device in men that closely mimics relevant clinical situations.3-7 In this model, native blood is drawn from healthy volunteers through a parallel-plate chamber device where blood components interact with collagen at well-established flow conditions. Blood flow conditions mimic wall shear rates as encountered in medium-sized (650 s1) and moderately stenosed small (2600 s1) arteries. This model has been used to investigate numerous antithrombotic strategies, and the results appear consistent with clinical data. Four polymorphisms were investigated. They were the variable number of tandem repeats (VNTRs) of GPIb, the 5T/C polymorphism in the Kozak sequence of GPIb, the 807C/T polymorphism of GPIa, and the PlA1/PlA2 polymorphism of GPIIb-IIIa.8-11     Study design Top Abstract Introduction Study design Results and discussion References Subjects The study population consisted of 40 healthy Caucasian male volunteers aged 21 to 41 years (mean ± 1 SD, 25 ± 4). They had no history or clinical signs of any disease. Clinical chemistry, hematologic, and hemostatic laboratory values were within the normal ranges. All subjects gave written, informed consent to the protocol, which was approved by the local Human Subjects Committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Toulouse). The volunteers were requested to come to the study center between 9:00 and 10:00 AM after 12 hours of fasting. They were not taking any medication known to affect blood coagulation or platelet function during the 10 days preceding the blood donations. In addition, all volunteers were nonsmoking subjects or subjects smoking less than 10 cigarettes/day, and they did not smoke on the day of the perfusion experiments. We have previously shown that smoking does not affect platelet thrombus formation in these conditions.12,13 Their body mass index was less than 25 kg/m.2 Genotyping of the 4 polymorphisms Genomic DNA was isolated from peripheral blood mononuclear cells, and the polymorphisms were determined by genomic polymerase chain reaction according to methods previously described.8-11 Preparation of thrombogenic surface Human type I collagen purified from pepsin-digested placenta collagens (Sigma, Saint-Quentin-Fallavier, France) was used. A fibrillar collagen suspension was obtained by dialysis at 4°C against 20 mM Na2HPO4, pH7.5, for 24 hours. It was spray-coated onto Thermanox plastic coverslips (Miles Laboratories, Naperville, IL) to a final density of 5 µg/cm.2 The coverslips were stored at room temperature for 15 to 20 hours before use.3 Perfusion experiments Perfusion experiments were performed with a parallel-plate perfusion chamber device at 37°C.3,4 Following blood sample collection, native blood was drawn directly from an antecubital vein of the volunteers by a 19-gauge infusion set (Ohmeda, Helsingborg, Sweden) over the collagen-coated coverslip positioned in the parallel-plate perfusion chamber. The blood flow rate was maintained at 10 mL/minute by a peristaltic roller pump (Minipuls, Gilson, Villiers-Le-Bel, France) placed distal to the chamber. The wall shear rates were 650 and 2600 s1. The blood perfusion experiment lasted for 4 minutes and was followed by a 30-second perfusion of phosphate-buffered saline at the same flow rate to wash out blood from the flow channel of the perfusion chamber. Subsequently, the coverslip covered by thrombotic deposits was placed in a plasmin solution and processed as previously described.5 Immunologic determination of platelet deposition Platelet deposition was quantified by measurement of a specific platelet  granule membrane protein, P-selectin.5 After centrifugation of the plasmin-digested thrombus, the pellet was dissolved in 400 µL lytic buffer, frozen and thawed 3 times, and then sonicated (4°C, 20 KHz) for 270 seconds. All samples of dissolved pellets were stored at 80°C until assayed for P-selectin by immunoenzymoassay (Bender MedSystems, Vienna, Austria). Results were expressed as the number of platelets deposited/cm2 (×107/cm2). Other laboratory procedures von Willebrand factor plasma levels were measured immunologically, using the Laurell method (Assera-vWf; Stago). Fibrinogen plasma levels were measured by the von Clauss method on an STA automate (Stago, Asnières, France). Hematocrit, white blood cell, and platelet counts were measured by an electronic counting device (Model S plus; Coulter Electronics, Hialeah, FL). Statistical analysis Results were expressed as the mean ± 1 SD. The relationship between the 4 polymorphisms and platelet deposition was analyzed by using a stepwise multiple linear regression analysis. This relationship was determined by adjusting other important blood parameters susceptible to influence platelet thrombus formation (ie, individual platelet count, white blood cell count, hematocrit, fibrinogen, and von Willebrand factor plasma levels). Two group comparisons were performed by using the nonparametric Mann-Whitney U test. All statistical tests of hypothesis were 2-tailed and were performed at .05 level of significance.     Results and discussion Top Abstract Introduction Study design Results and discussion References The characteristics of platelet thrombus formation in blood from volunteers with the 4 polymorphisms are shown in Table 1. Thrombi formed on collagen at intermediate and high shear rates were essentially composed of platelets with a few fibrin deposits.3 Platelet thrombus formation increased with increasing shear rate. At 650 s1 platelet deposition was significantly affected by the Kozak sequence polymorphism of GPIb and was significantly lower in T/T than in T/C subjects (P = .01). None of the other polymorphisms evaluated in this study affected platelet thrombus formation. At 2600 s1 platelet thrombus formation was significantly affected only by the 807C/T polymorphism of the collagen receptor (P = .03) GPIa; individuals homozygous for the 807C allele had significantly lower platelet deposits than those homozygous for the 807T allele (P = .03, Mann-Whitney U test). The VNTR polymorphism of GPIb and the PlA1/PlA2 polymorphism of GPIIb-IIIa did not affect platelet deposition.                                View this table: [in this window] [in a new window]   Table 1. Relationships between platelet membrane glycoprotein polymorphisms and collagen-induced thrombus formation Perfusion times were 4 minutes. Because the 807C/T polymorphism has been shown to influence the rate of platelet adhesion,14 it is possible that the influence of this polymorphism or of the Kozak polymorphism would have been better observed if platelet deposition had been measured at earlier time points. Interestingly, the 2 polymorphisms found to modulate platelet thrombus formation (ie, the 5T/C polymorphism in the Kozak sequence of GPIb and the 807C/T polymorphism of GPIa) appear to influence the number of receptor copies on the platelet surface. The Kozak sequence polymorphism has been shown to be a determinant of the surface density of the platelet GPIb receptor, and the allele C has been associated with increased expression of this receptor.15 The 807C allele of the GPIa gene has been associated with the lowest GPIa receptor density and the 807T allele with high GPIa receptor density.14 We suggest that the increase in the density of these platelet receptors may predispose the platelets to adhere more readily to the thrombogenic surface and, therefore, may augment the thrombotic tendency, notably at high shear rate. One should note that the 807C/T and Kozak polymorphisms were not linked (NS, Fisher test) and that there was no interaction between these 2 polymorphisms. In conclusion, our results support the clinical studies reporting a positive association between the Kozak and 807C/T polymorphisms and acute arterial thrombotic events. In addition, our studies may explain at least some of the controversies reported between these genetic variations and the thrombotic phenotype, because the effect of this polymorphism on platelet thrombus formation depends on the local blood flow conditions. The potential effect of this polymorphism on arterial thrombogenesis must be given consideration, because fibrillar collagens are major components of the subendothelial matrix that trigger thrombus formation.     Footnotes Submitted May 2, 2001; accepted July 5, 2002. Supported by research grants from the Délégation Régionale à la Recherche Clinique (No 98-17-L, CHU Purpan, Toulouse, France) and from INSERM/MERCK, SHARP, DOME, and CHIBRET (No. 98 CIC TO 07). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Yves Cadroy, Laboratoire de Recherche sur l'Hémostase et la Thrombose, Pavillon Lefèbvre, CHU Purpan, 31059 Toulouse, France; e-mail: cadroy.y{at}chu-toulouse.fr.     References Top Abstract Introduction Study design Results and discussion References 1. Ridker PM, Stampfer MJ. Assessment of genetic markers for coronary thrombosis: promise and precaution. Lancet. 1999;353:353-354. 2. Lane DA, Grant PJ. Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood. 2000;95:1517-1532[Free Full Text]. 3. Sakariassen KS, Joss R, Muggli R, et al. Collagen type III induced ex vivo thrombogenesis in humans: role of platelets and leucocytes in deposition of fibrin. Arteriosclerosis. 1990;10:276-284[Abstract/Free Full Text]. 4. Diquélou A, Lemozy S, Dupouy D, Boneu B, Sakariassen KS, Cadroy Y. Effect of blood flow on thrombin generation is dependent on the nature of thrombogenic surface. Blood. 1994;84:2206-2213[Abstract/Free Full Text]. 5. Bossavy JP, Sakariassen KS, Barret A, Boneu B, Cadroy Y. A new method for quantifying platelet deposition in flowing native blood in an ex vivo model of human thrombogenesis. Thromb Haemostas. 1998;79:162-168[Medline] [Order article via Infotrieve]. 6. Bossavy JP, Thalamas C, Sagnard L, et al. A double-blind randomized comparison of combined aspirin and ticlopidine therapy versus aspirin or ticlopidine alone on experimental arterial thrombogenesis in man. Blood. 1998;92:1518-1525[Abstract/Free Full Text]. 7. Cadroy Y, Bossavy JP, Thalamas C, Sagnard L, Sakariassen KS, Boneu B. Early potent antithrombotic effect with combined aspirin and a loading dose of clopidogrel on experimental arterial thrombogenesis in man. Circulation. 2000;101:2823-2828[Abstract/Free Full Text]. 8. Carter AM, Catto AJ, Bamford JM, Grant PJ. Platelet GPIIIa PlA and GPIb variable number tandem repeat polymorphisms and markers of platelet activation in acute stroke. Arterioscler Thromb Vasc Biol. 1998;18:1124-1131[Abstract/Free Full Text]. 9. Kaski S, Kekomäki R, Partanen J. Systematic screening for genetic polymorphism in human platelet glycoprotein Ib alpha. Immunogenetics. 1996;44:170-176[CrossRef][Medline] [Order article via Infotrieve]. 10. Di Paola J, Federici AB, Mannucci PM, et al. Low platelet 21 levels in type I von Willebrand disease correlate with impaired platelet function in a high shear stress system. Blood. 1999;93:3578-3582[Abstract/Free Full Text]. 11. Weiss EJ, Bray PF, Tayback M, et al. A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med. 1996;334:1090-1094[Abstract/Free Full Text]. 12. Roald HE, Orvim U, Bakken IJ, Barstad RM, Kierulf P, Sakariassen KS. Modulation of thrombotic responses in moderately stenosed arteries by cigarette smoking and aspirin ingestion. Arterioscler Thromb. 1994;14:617-621[Abstract/Free Full Text]. 13. Roald HE, Lyberg T, Dedichen H, et al. Collagen-induced thrombus formation in flowing non-anticoagulated human blood from habitual smokers and non-smoking patients with severe peripheral atherosclerotic disease. Arterioscler Thromb Vasc Biol. 1995;15:218-223. 14. Kritzik B, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ. Nucleotide polymorphisms in the 2 gene define multiple alleles that are associated with differences in platelet 21 density. Blood. 1998;92:2382-2388[Abstract/Free Full Text]. 15. Afshar-Khargan V, Li CQ, Khoshnevis-Asl M, Lopez JA. Kozak sequence polymorphism of the glycoprotein (GP) Ib gene is a major determinant of the plasma membrane levels of the platelet GP Ib-IX-V complex. 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	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020