Changes in platelet membrane glycoproteins and platelet-leukocyte interaction during hemodialysis

Platelet aggregation and interaction with other vascular cells play a key role in hemostatic events during hemodialysis. We studied seven patients with end-stage renal failure on long-term hemodialysis treatment. Flow-cytometric techniques and platelet-specific monoclonal antibodies were used to measure the platelet surface expression of glycoproteins - fibrinogen receptor on glycoprotein IIb-IIIa (GP IIb-IIIa; CD41) and -granule membrane protein (GMP-140; CD62). In addition, adhesion of platelets or platelet microparticles with leukocytes was evaluated by appearance of the platelet-specific antigen (GP IIb-IIIa) on leukocytes. Blood samples were taken before the start of dialysis and 15, 60, and 240 min thereafter. There was a significant increase in fibrinogen receptor activation on circulating platelets after 15 min of dialysis treatment (P < 0.001) and enhanced degranulation of GMP-140 (P < 0.05). In parallel, the interaction of platelets with neutrophils and monocytes also increased with the duration of dialysis and was maximal after 15 min (P < 0.001). We conclude that the platelet fibrinogen receptor on GP IIb-IIIa in circulating platelets is activated during hemodialysis and is associated with increased adhesion of platelets or platelet microparticles with circulating leukocytes. Thus, the phenomenon described here of platelet-leukocyte interaction could be pathophysiologically important for the development of dialysis-associated leukopenia.Abbreviations GP IIb-IIIa glycoprotein IIb-IIIa - GMP140 granule membrane protein 140 - LIBS ligand-induced binding site - FACS flow-activated cell sorting - mAb monoclonal antibody Correspondence to: M. Gawaz     

Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation

The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm² for 1 min at 37°C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin α_IIbβ₃) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm², whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin α_vβ₃) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm²; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same β₃ subunit as GP IIb-IIIa, plays no role in SIPA. On the basis of these results, the assessment of platelet size distributions provides a sensitive and quantitative measurement for the study of SIPA.     

中华眼镜蛇毒F组分抗血小板聚集的作用机制

目的探讨中华眼镜蛇毒F组分抑制血小板聚集的作用机制。方法用比浊法测定中华眼镜蛇毒F组分对二磷酸腺苷、花生四烯酸和血小板活化因子诱导血小板聚集作用的影响,流式细胞术观察中华眼镜蛇毒F组分对荧光标记的单克隆抗体CD41(FITC-CD41)和CD61(FITC-CD61)与血小板膜糖蛋白IIb/IIIa(GPIIb/IIIa)结合的影响。结果中华眼镜蛇毒F组分明显抑制二磷酸腺苷、花生四烯酸和血小板活化因子诱导的血小板聚集,其作用呈现一定程度的剂量依赖关系。中华眼镜蛇毒F组分可以明显降低单克隆抗体CD41(抗GPIIb)与血小板的结合率,而对单克隆抗体CD61(抗GPIIIa)与血小板的结合率没有影响。结论中华眼镜蛇毒F组分可以抑制多种激动剂诱导的血小板聚集,其机制和中华眼镜蛇毒F组分与血小板膜糖蛋白IIb/IIIa复合物的结合有关。     

中华眼镜蛇毒F组分抗血小板聚集的作用机制

目的 探讨中华眼镜蛇毒F组分抑制血小板聚集的作用机制.方法 用比浊法测定中华眼镜蛇毒F组分对二磷酸腺苷、花生四烯酸和血小板活化因子诱导血小板聚集作用的影响,流式细胞术观察中华眼镜蛇毒F组分对荧光标记的单克隆抗体CD41(FITC-CD41)和CD61(FITC-CD61)与血小板膜糖蛋白Ⅱb/Ⅲa(GPⅡb/Ⅲa)结合的影响.结果 中华眼镜蛇毒F组分明显抑制二磷酸腺苷、花生四烯酸和血小板活化因子诱导的血小板聚集,其作用呈现一定程度的剂量依赖关系.中华眼镜蛇毒F组分可以明显降低单克隆抗体CD41(抗GPⅡb)与血小板的结合率,而对单克隆抗体CD61(抗GPⅢa)与血小板的结合率没有影响.结论 中华眼镜蛇毒F组分可以抑制多种激动剂诱导的血小板聚集,其机制和中华眼镜蛇毒F组分与血小板膜糖蛋白Ⅱb/Ⅲa复合物的结合有关.     

The platelet Fc receptor,FcγRIIa

Human platelets express FcγRIIa, the low-affinity receptor for the constant fragment (Fc) of immunoglobulin (Ig) G that is also found on neutrophils, monocytes, and macrophages. Engagement of this receptor on platelets by immune complexes triggers intracellular signaling events that lead to platelet activation and aggregation. Importantly these events occur in vivo, particularly in response to pathological immune complexes, and engagement of this receptor on platelets has been causally linked to disease pathology. In this review, we will highlight some of the key features of this receptor in the context of the platelet surface, and examine the functions of platelet FcγRIIa in normal hemostasis and in response to injury and infection. This review will also highlight pathological consequences of engagement of this receptor in platelet-based autoimmune disorders. Finally, we present some new data investigating whether levels of the extracellular ligand-binding region of platelet glycoprotein VI which is rapidly shed upon engagement of platelet FcγRIIa by autoantibodies, can report on the presence of pathological anti-heparin/platelet factor 4 immune complexes and thus identify patients with pathological autoantibodies who are at the greatest risk of developing life-threatening thrombosis in the setting of heparin-induced thrombocytopenia.     

Effects of α<Subscript>1</Subscript>-acid glycoprotein on hemostasis in experimental septic peritonitis

Pathogenesis of hemostasis disorders in septic peritonitis and the possibility of their correction with acute phase protein (α₁-acid glycoprotein; two doses of 150 mg/kg) were experimentally studied on outbred albino rats. Platelets count in the peripheral blood and their adhesion to endothelium did not change during peritonitis, while aggregation activity increased due to increased rate and shorter time of aggregation, which was associated with the development of hypercoagulation involving the intrinsic and common coagulation pathways and reduction of antithrombin activity. α₁-Acid glycoprotein increased platelet count above the normal level, normalized aggregation rate, some blood clotting parameters, and antithrombin activity. Hence, α₁-acid glycoprotein is a polyfunctional protein modulating all pathogenetic components in the development of blood clotting disorders during septic peritonitis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 143–145, August, 2007     

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.     

脑梗死急性期患者血小板膜糖蛋白Ia C807T基因多态性和血小板功能

目的:探讨血小板膜糖蛋白(GP)Ia C807T基因多态性及与脑梗死急性期患者血小板功能的关系。方法:应用聚合酶链反应限制性片段长度多态性(PCR-RELP)方法检测97例急性期脑梗死患者(脑梗死组)及99例正常对照者(对照组)的GPIa C807T基因型,采用全血流式细胞术检测血小板P选择素(CD62P)表达率,采用比浊法检测花生四烯酸、二磷酸腺苷诱导的血小板最大聚集率(MARAA、MARADP)。结果:脑梗死组GPIa T等位基因频率高于对照组,差异有统计学意义(χ2=5.369,P<0.05);脑梗死组CD62P表达率、血小板MARAA、MARADP高于对照组(P<0.05)。对照组TT+CT基因型CD62P表达率和MARADP高于CC基因型(P<0.05);脑梗死组TT+CT基因型CD62P表达率和MARAA、MARADP与CC基因型差异无统计学意义(P>0.05)。结论:GPIaC807T可能是脑梗死发病的遗传危险因素。血小板活化和聚集功能增强可能是T等位基因促进脑梗死发病的机制之一。     

Clinical spectrum of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia is a well defined inherited disorder of platelet function characterized by qualitative and qualitative defect in cytoadhesive membrane protein, glycoprotein IIb-IIIa (the platelet fibrinogen receptor). From January 1990 to October, 1999, five patients who presented with mucocutaneous bleeding were detected to have Glanzmann's thrombasthenia. Clinical and laboratory spectrum of this rare disorder was studied which revealed heterogeneity of disease with respect to nature and severity of bleeding unpredictable by laboratory findings.     

血管性血友病因子和血小板表面受体突变的数据库构建及数据分析

背景：血管性血友病因子和血小板表面受体之间的相互作用在血小板黏附、传播、聚集等血栓形成过程中起到关键作用。目前关于血管性血友病因子突变信息的数据库并不完善且血小板表面受体相关的数据库仍未构建。目的：致力于构建血管性血友病因子及血小板糖蛋白Ibα相关突变的数据库,以利于相关领域的研究人员快速查找到该分子对的重要突变信息。方法：以数据库Uniprot、VWFdb及文献报道的突变信息为数据源,采用MySQL和Apache为后台数据库和服务器,运用PHP语言开发血管性血友病因子及血小板糖蛋白Ibα突变数据库。结果与结论：收集了341条血管性血友病因子,13条血小板糖蛋白Ibα的野生或者突变序列数据,并人工构建A1结构域的虚拟突变3 920条,建立了包括背景介绍、相关病理图册和资料下载等登录页面,初步实现了数据检索、突变位点分析以及虚拟突变位点信息等相关功能。该数据库较全面搜集及分析血管性血友病因子及血小板糖蛋白Ibα的数据信息,有利于研究人员对血管性血友病因子和血小板糖蛋白Ibα相关信息的查询,有助于新型抗血栓药物的研发,进一步提高临床诊断和治疗水平。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa.

Platelet surface immunoglobulin G (PSIgG) was measured ex vivo in nine patients with stable angina pectoris receiving continuous (48-96 h) infusions of Fab fragments of a chimaeric MoAb (human IgG with murine variable regions) to platelet glycoprotein IIb-IIIa. PSIgG was measured using flow cytometry (FC) and an Fc-specific anti-IgG polyclonal antibody, which did not cross-react with the chimaeric Fab fragment (c7E3-Fab). A variable but statistically significant (P < 0.05) elevation of PSIgG was present within 24 h after the onset of the infusion, and was more marked (P < 0.01) several days after the end of the infusion despite an exponential fall in platelet surface c7E3-Fab post-infusion. PSIgG returned to normal within 2 weeks after the end of the infusion. The timing of IgG recruitment to the platelet surface suggested the pre-existence in the patients' plasma of IgG binding to c7E3-Fab-bearing platelets. None of the patients developed thrombocytopenia. In order to assess the incidence of IgG bindable to c7E3-Fab-bearing platelets in controls clinically comparable to the c7E3-Fab infusion patients, normal platelets coated with either chimaeric (c) or murine (m) 7E3-Fab were incubated with plasmas from 21 patients with ischaemic heart disease, and recruitment of IgG to the platelet surface was measured by FC. Fourteen of the 21 plasmas contained IgG bindable to c7E3-Fab-coated platelets, whereas only one of the 21 plasmas contained IgG bindable to m7E3-Fab-coated platelets (a highly significant difference, P < 0.001). These findings indicate that infusions of Fab fragments of the chimaeric anti-platelet antibody 7E3 are often associated with elevations in PSIgG, which are probably due to pre-existing 'naturally occurring' antibodies to the Fab fragments of chimaeric (but not murine) 7E3, and most probably other chimaeric MoAbs. The possible clinical significance of such ex vivo measured activities is at present a matter for speculation, and requires further study.     

New strategy of platelet substitutes for enhancing platelet aggregation at high shear rates: cooperative effects of a mixed system of fibrinogen γ-chain dodecapeptide- or glycoprotein Ibα-conjugated latex beads under flow conditions

To construct platelet substitutes that have hemostatic properties over a wide range of shear rates, we used fibrinogen γ-chain carboxy-terminal sequence HHLGGAKQAGDV (H12), which recognizes activated platelets at low shear rates, and a recombinant water-soluble moiety of the platelet glycoprotein (rGPIbα), which recognizes von Willebrand factor at high shear rates. Three kinds of samples were prepared for this purpose: H12-conjugated latex beads (H12-latex beads), rGPIbα-latex beads, and H12/rGPIbα-latex beads. These samples were evaluated in thrombocytopenia-imitation blood at various flow conditions. Based on ADP-induced platelet aggregation studies, the H12-latex beads significantly enhanced platelet aggregation via H12 binding with GPIIb/IIIa activated on the surface of activated platelets, whereas the rGPIbα-latex beads did not support platelet aggregation. In the case of the H12/rGPIbα-latex beads, the function of H12 was suppressed by steric hindrance from the larger rGPIbα bound to the latex bead. A mixture of the H12-latex beads and the rGPIbα-latex beads adhered to a collagen surface over a wide range of shear rates. In particular, at high shear rates, a cooperative effect was observed in the enhancement of platelet thrombus formation compared with H12-latex beads or rGPIbα-latex beads alone. We propose that a mixed system of H12- and rGPIbα-conjugated nanoparticles is a more effective platelet substitute than each of the beads used alone and has enhanced platelet aggregation properties.     

Platelet pro-aggregatory effects of CD40L monoclonal antibody

An unexpected high incidence of thromboembolic complications has been described in patients with systemic autoimmune diseases treated with CD40L immunotherapy. Since activated platelets express CD40L, we aimed to investigate the effects of CD40L mAb in platelet aggregation induced by physiological stimuli. Optical aggregometry was performed on platelet-rich plasma and washed platelets obtained from systemic venous blood (0.38% citrate) of anesthetized pigs. CD40L mAb clone 5c8, used in clinical trials for autoimmune diseases, was used. In platelet-rich plasma, CD40L mAb neither induced platelet aggregation per se, nor significantly affected maximal aggregation or slope of ADP-induced aggregation curves. However, it dose-dependently inhibited spontaneous deaggregation observed in ADP-stimulated samples. This effect was not observed with an irrelevant isotype-matched immunoglobulin. The stabilizing effect on platelet aggregates was neither glycoprotein IIb/IIIa-mediated nor Ca2+-dependent but was abolished by acetylsalicylic acid pretreatment. F(ab')2 fragments did not stabilize ADP-induced platelet aggregates but inhibited the stabilizing effect of CD40L mAb. Similar results were obtained with washed platelets, although higher amplification of ADP-induced aggregation was observed. In conclusion, CD40L expression produced by physiological or pathophysiological platelet activation can sustain a pro-aggregatory effect of CD40L mAb by a mechanism involving mAb Fc domain. These results could help to explain the mechanism of CD40L mAb-induced thromboembolic complications.     

抗血小板糖蛋白VI单链抗体的制备和功能研究

将抗血小板膜表面糖蛋白VI(GPVI)单克隆抗体SZ118构建成单链抗体,以研究开发新型抗血栓药物。采用RT-PCR技术,从分泌SZ118杂交瘤细胞中克隆该抗体的重链可变区(VH)和轻链可变区(VL)基因。经测序鉴定后构建表达质粒pET20b(+)-SZ118scFv,并体外表达SZ118单链抗体(SZ118scFv)。同时测定了SZ118scFv对血小板黏附和聚集功能的影响。结果表明成功克隆了SZ118 VH和VL基因并正确构建了SZ118scFv表达质粒。复性后SZ118scFv保留源抗体与血小板结合能力,显著抑制纤维状胶原和Convulxin诱导的血小板聚集,且呈剂量依赖性,最大抑制率分别为(84.3%±5.6%)和(50.3%±15.5%);SZ118scFv明显抑制高剪切力条件下血小板与纤维状胶原的黏附,抑制率达68.3%。结果提示,成功制备了SZ118scFv,SZ118scFv可阻断血小板与胶原的相互作用,具有潜在的实际应用价值。     

血小板糖蛋白GPIb α Kozak基因多态性与短暂性脑缺血发作的相关性研究

目的研究青岛地区汉族人群血小板糖蛋白（platelet glycoprotein，GP）GPIb α Kozak序列-5T／C基因多态性与短暂性脑缺血发作（transient ischemic attack，TIA）的关系。方法应用序列特异性引物PCR扩增和基因序列测定技术检测90例TIA患者和80名对照组的GPIb α Kozak序列-5T／C基因多态性，比较各基因型的表达与TIA的关系。结果TIA组CC基因型频率为0．166，明显高于对照组（0．075），两组差异有统计学意义（P〈0．05），TIA组C等位基因与对照组比较显著增高（P〈0．05）。Logistic回归分析结果显示C等位基因与TIA发病正相关（P=0．016）。结论血小板GPIb α Kozak基因多态性与TIA相关，CC基因型和C等位基因可能是TIA的危险因素。     

Human monoclonal autoantibody 2E7 is specific for a peptide sequence of platelet glycoprotein IIb. Localization of the epitope to IIb231-238 with an immunodominant Trp235.

2E7 is a human monoclonal IgM autoantibody that binds to a site on the heavy chain of the human platelet integrin alpha subunit glycoprotein IIb. The epitope recognized by 2E7 is stable to denaturation with sodium dodecyl sulfate and reduction of disulfide bonds but is destroyed by proteolysis with papain, chymotrypsin or elastase. By evaluating the reaction of 2E7 with a number of protein sequences from the IIb heavy chain, we have determined that the epitope is located in the octapeptide Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser (FDGYWGYS), corresponding to residues 231-238, and that substitution of the Trp at position 235 completely destroys the epitope. This represents the first precise localization of an epitope on the human platelet integrin IIb-IIIa or on any platelet membrane glycoprotein that is recognized by a human autoantibody.     

Antibody binding to platelet antigens in acute and chronic idiopathic thrombocytopenic purpura: a platelet membrane ELISA for the detection of antiplatelet antibodies in serum.

A solid-phase platelet membrane enzyme linked immunosorbent assay (ELISA) was developed for the detection of circulating platelet binding IgG in serum. 18 out of 28 (64%) sera from patients with chronic idiopathic thrombocytopenic purpura (ITP) and 10 out of 21 (48%) sera from patients with acute ITP contained more platelet binding IgG than the mean +2 s.d. of 25 normal control sera. IgG-F(ab')2 fragments from two anti-PlA1 sera, from six out of seven positive chronic ITP sera and from four out of eight positive acute ITP sera bound to normal platelet membrane. When IgG-F(ab')2 preparations, which reacted with normal membrane, were tested against glycoprotein IIb-IIIa-deficient platelet membrane obtained from a patient with Glanzmann's thrombasthaenia, the reactions of F(ab')2 fragments from two anti-PlA1 sera, from three out of four acute ITP sera and from four out of six chronic ITP sera were reduced to control levels. It is concluded that IgG-F(ab')2 antibody binding to platelet specific antigens, some of which are associated with the glycoprotein IIb-IIIa complex, can be demonstrated not only in anti-PlA1 sera or sera from chronic ITP patients but also in sera from some patients with acute ITP. Moreover, when the IgG binding to platelet membrane of whole sera is tested, positive results may sometimes be ascribed to the presence of immune complexes.     

Die temperaturabhängige Spontanaggregation der Thrombozyten bei Polycythaemia vera und primärer Thrombozythaemie: Ein diagnostisches Kriterium

Summary In twenty patients suffering from myeloproliferative syndromes (nine with polycythaemia vera, 11 with primary thrombocythaemia) platelet aggregation was tested following incubation of blood samples at 4° C, room temperature, 30° C and 37° C. A spontaneous platelet aggregation following incubation at room temperature took place in 65% of patients with primary thrombocythaemia. At the two higher temperatures, positive results were seen in 72% and 92% of cases, respectively. Seven of nine patients (78%) showed a pathological spontaneous platelet aggregation at room temperature. Pathological spontaneous platelet aggregation at 30° C occurred in eight of nine (89%) cases. The spontaneous platelet aggregation test is a simple method to diagnose primary thrombocythaemia and asymptomatic polycythaemia vera, the sensitivity of which increases considerably following incubation of blood samples at two and three different temperatures.Mit Unterstützung der EMDO-Stiftung     

人类血小板同种抗原相关的膜糖蛋白基因多态性研究

目的 探讨人类血小板同种抗原(human platelet alloantigen,HPA)-1～17w相关的血小板膜糖蛋白基因多态性.方法 以自行设计的PCR引物特异性扩增HPA-1～17w相关的血小板膜糖蛋白基因片段,PCR产物经酶切纯化后直接进行DNA序列分析,根据序列特征确定HPA基因型和相应膜糖蛋白基因多态性.结果 通过对112名随机汉族个体的糖蛋白基因序列分析,发现13个新的单核昔酸多态性位点和1个微卫星重复序列.其中ITGB3基因上的2个变异位点1333G/A和1960G/A引起膜糖蛋白GPⅢa的V419M和E628K氨基酸改变.结论 膜糖蛋白基因具有多态性位点,可能引起膜糖蛋白结构的改变,同时可能影响现有部分HPA基因分型方法的准确性.

Abstract：

Objective To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w. Methods The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author' s designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms. Results Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/Aand 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPⅢ a.Conclusion New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.     

Factors influencing platelet aggregation in whole blood

In order to evaluate the interference of blood cells on platelet aggregation, spontaneous platelet aggregation (SPA), ADP, and collagen-induced platelet aggregation were investigated in whole blood by the impedance method and in platelet-rich plasma (PRP) by densitometric and impedance aggregometers. Stirring of the sample induced a significant decrease of neutrophils (P less than 0.001) but no changes of red blood cell (RBC) and platelet count. After collagen addition, a further decrease of neutrophils was observed, while RBC count was unmodified. The occurrence of SPA was not different in whole blood and in PRP. Platelet number and hematocrit did not affect either spontaneous or collagen-induced aggregation. A significant linear relation (r = -0.60, P less than 0.01) between neutrophil count and collagen whole blood platelet aggregation was found. Collagen- and ADP-induced aggregation were significantly higher and lower, respectively, in whole blood than in PRP using the densitometric method. No differences were observed in SPA and collagen platelet aggregation according to age and sex.