Platelets adhere to sulfatides by von Willebrand factor dependent and independent mechanisms

Unstimulated human platelets from normal volunteers adhere to sulfatides (galactosylceramide-I3-sulfate) as single cells but do not adhere appreciably to other lipids including gangliosides, neutral glycolipids, phospholipids or cholesterol-3-SO4. Platelet adhesion to sulfatide is saturable and dose-dependent, reaches maximal levels in 90 to 120 min, and is not divalent cation-dependent. Because sulfatides bind von Willebrand factor (vWf) with specificity and high affinity and platelet adhesion to structurally related sulfated glycolipids is approximately proportionate to their ability to bind vWf, we examined whether vWf mediates platelet adhesion to sulfatides. Platelets from a patient with severe Type I von Willebrand's disease adhere poorly to sulfatides. However, adhesion to levels seen with normal platelets is restored by the addition of vWf. Adhesion of normal platelets can be partially inhibited by a monospecific antibody to vWf. Normal platelet adhesion to sulfatides, however, is not increased following preincubation with vWf. Both vWf binding and platelet adhesion to sulfatides can be inhibited by the sulfated polysaccharide dextran sulfate at low concentration, fucoidan at high concentrations, but not by heparin, fibrinogen, fibronectin, or the synthetic peptides Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro. Thus, adhesion to sulfatides appears to be of two types; vWf dependent (50-75%) and vWf independent (25-50%).     

Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor

Recently, we described a method of testing platelet deposition on extracellular matrix under flow conditions. The method was used for assessment of platelet function in various platelet disorders, for monitoring of replacement and anti-platelet therapy. In the present study, we investigated platelet deposition on a polystyrene surface compared with that on extracellular matrix, under defined shear rates, using the original Cone and Plate(let) Analyzer. A correlation of adhesion rate (surface coverage) and aggregate formation (average size) of platelets from normal citrated blood between polystyrene and extracellular matrix was observed. Blocking of von Willebrand factor binding to glycoprotein Ib by a recombinant von Willebrand factor fragment substantially decreased platelet adhesion to both surfaces. Blocking of GPIIb-IIIa by Arg-Gly-Asp-Ser peptide prevented platelet adhesion to the polystyrene while an extensive adhesion of single platelets to extracellular matrix was observed. Furthermore, platelet adhesion to polystyrene but not to extracellular matrix was completely inhibited by platelet inactivation with prostaglandin E(1). Platelets from patients with severe von Willebrand disease yielded very low adhesion to both polystyrene and extracellular matrix. The addition of von Willebrand factor to the blood of these patients or pre-coating of polystyrene surface with von Willebrand factor restored the ability of platelets to adhere and aggregate on the surface. Platelets from patients with Glanzmann's thrombasthenia and afibrinogenemia adhered to extracellular matrix (with defective aggregate formation), while they failed to adhere to the polystyrene. Fibrinogen added to afibrinogenemia blood or pre-coating of the polystyrene with fibrinogen restored the ability of platelets to adhere and aggregate on the surface. In conclusion, the polystyrene surface, like extracellular matrix, can be used to assess platelet function disorders taking in account that platelet deposition on polystyrene under flow is absolutely dependent on platelet activation and on the presence of fibrinogen, von Willebrand factor, and their receptors.     

Real-time analysis of the interaction of platelets with immobilized thrombospondin under flow conditions

The platelet granule protein (TS) is extracellularly secreted upon platelet activation and then binds to the platelet surface where it can interact with various adhesive proteins. Here, we have analyzed platelet interactions with a TS-coated surface under flow conditions, a model for platelet adhesion onto surface-bound TS under physiological conditions. Platelets exhibited temporary, very short-time adhesion on the TS surface, but no firm adhesion. This adhesion was inhibited by NNKY5-5 (anti-glycoprotein (GP) Ib antibody) and AJvW-2 (anti-von Willebrand factor (vWF)), indicating that both platelet GP Ib and plasma vWF contribute to this interaction. Antibodies against platelet collagen receptor integrin ₂β₁ had no significant effect. These results suggested that binding of vWF to TS is the first step in platelet interaction with the TS surface. By surface plasmon resonance spectroscopy, a dissociation constant (K_d) of 3.97×10⁻⁷ M was obtained for the binding reaction between immobilized TS and vWF. These results suggest the following model for platelet interaction with the TS surface under flow: plasma vWF first binds to the immobilized TS and then platelets interact with the TS-bound vWF. A low density of bound vWF would account for the observed weak interaction between TS and platelets under flow.     

Characterisation of a monoclonal antibody to von Willebrand factor as a potent inhibitor of ristocetin-mediated platelet interaction and platelet adhesion

We studied a murine monoclonal antibody (211 A6) to von Willebrand factor (vWF) with a view to investigating structure-relationship of plasma vWF. The specificity of this antibody has been substantiated by ELISA tests and indirect immunofluorescence. It reacts with purified vWF, normal plasma but not with plasma or platelets from a severe von Willebrand's disease patient. Monoclonal antibody 211 A6 is a potent inhibitor of ristocetin-induced platelet aggregation. The 125I-FVIII/vWF binding to platelets in presence of ristocetin is totally inhibited by low 211 A6 concentrations. Thrombin-induced binding of vWF to platelets is not affected by 211 A6. The ability of this antibody to inhibit platelet adhesion to subendothelium and to collagen was investigated with a perfusion model. The complete inhibition of platelet adhesion by 211 A6 questions the similarity or the interrelationship in vWF domains involved in ristocetin-induced platelet functions and platelet adhesion.     

Studies on the factor VIII/von Willebrand factor antigen on human platelets

A purified protein having both Factor VIII activity (coagulant assay) and von Willebrand factor activity (platelet retention and ristocetin aggregation assays) was used to immunize a goat. The resulting antiserum neutralized Factor VIII coagulant activity, decreased platelet retention of normal blood and blocked ristocetin aggregation of normal platelet rich plasma.

This same antiserum acted as an “anti-platelet” antibody in serotonin release, platelet aggregation and immunofluorescence with normal, hemophilic and von Willebrand platelets. However, after suitable absorption with small numbers of platelets this antiserum recognized Factor VIII/von Willebrand factor antigen only on normal and hemophilic platelets but not on platelets from two patients with severe von Willebrand's disease.

It is possible that antisera produced against Factor VIII purified from plasma not rendered completely free of platelets contains antibodies to platelet membrane material.     

A polyclonal antibody to protein disulfide isomerase induces platelet aggregation and secretion

Monoclonal mouse antiplatelet antibodies against a variety of platelet surface components can activate platelets, causing platelet aggregation and secretion. The mechanism involves binding of the Fab domain to a platelet surface antigen, and the activation occurs through an interaction of the Fc domain with the platelet FcγRII receptor. There is almost no information on FcγRII receptor-dependent activation of platelets by polyclonal rabbit antibodies. We presently report that a polyclonal rabbit antibody to a platelet surface antigen, protein disulfide isomerase, induces platelet aggregation and secretion. These effects are seen with concentrations of the antiprotein disulfide isomerase antibody as low as 25 to 40 μg/mL. Fab and F(ab′)₂ preparations of the rabbit antiprotein disulfide isomerase antibody do not cause aggregation. Fab made from the rabbit antiprotein disulfide isomerase antibody as well as a monoclonal antibody to the FcγRII (IV.3) receptor block the aggregation and secretion responses. Aggregation and secretion are inhibited by an antiglycoprotein IIbIIIa antibody, which blocks fibrinogen binding and wortmannin, an inhibitor of phosphoinositide 3-kinase. Aspirin, prostaglandin E₁, and Ethylenediaminetetraacetic acid (EDTA) also block the platelet responses. These data suggest that activation of platelets by polyclonal antibodies occurs by mechanisms similar to those found with activating monoclonal antibodies.     

Platelet adhesion to collagen in subtypes of type I von Willebrand's disease is dependent on platelet von Willebrand factor

Von Willebrand's disease type I, characterized by low levels of factor VIII coagulant activity (VIII: C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (RiCof) (1), can be subdivided on the basis of platelet von Willebrand factor into subtype platelet normal, platelet discordant, and platelet low (2). We have investigated the contribution of platelet von Willebrand factor in these various subtypes to platelet adhesion using the rectangular perfusion chamber of Sakariassen et al. (3) with fibrillar collagen or a fibroblast matrix as adhesive surfaces. Platelet adhesion to fibrillar collagen was decreased in all subtypes of von Willebrand's disease, but not as low as in severe von Willebrand's disease. A close correlation was observed between platelet adhesion to collagen and plasma vWF:Ag in severe von Willebrand's disease, subtype platelet low, subtype platelet discordant, and normal controls. The platelet adhesion in subtype platelet normal was higher than expected from the plasma vWF:Ag level. Perfusions in which washed platelets were added to a human albumin solution together with red blood cells gave similar adhesion values in subtype platelet normal and normal controls; adhesion was decreased in subtype platelet discordant, and the lowest values were found in subtype platelet low and in severe von Willebrand's disease. These data indicate that platelet von Willebrand factor may contribute to platelet adhesion, when plasma von Willebrand factor is low. Perfusion studies over a fibroblast matrix gave similar low adhesion values for subtype platelet low and platelet normal, indicating that the contribution of platelet von Willebrand factor can only be observed on a strongly activating surface such as fibrillar collagen.     

Aeguorin-detected calcium changes in stimulated thrombasthenic platelets. Aggregation - dependent calcium movement in response to ADP

Calcium changes in normal and thrombasthenic platelets were recorded using the PICA-apparatus. Aequorin was loaded in the presence of DMSO, EGTA and PGE1. Platelets of three patients with type I thrombasthenia stimulated with A-23,187, thrombin, PMA in the presence 1mM Ca⁺⁺ and 1mM Mg⁺⁺ were able to normally raise their calcium concentrations. The maximal values could be found below the normal range with collagen, ADP and PAF-acether. Calcium mobilization from internal stores in response to thrombin was normal. There were two calcium peaks in normal platelets stimulated with ADP. The second one was suppressed by omitting fibrinogen, stirring, or by adding aspirin, and was absent in thrombasthenic platelets. Thus the GP IIb-Illa complex is not a prerequisite for calcium fluxes but is involved, when weak agonists such ADP are used, through an aggregation-dependent reinforcement of platelet activation.     

Further studies on aggregation of platelet-type von Willebrand's disease platelets by human von Willebrand factor

Platelet-type von Willebrand's disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF-induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation.     

Baboon fibrinogen adsorption and platelet adhesion to polymeric materials

The role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode's buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and 111In radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma. When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well. Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i.e., serum, heat defibrinogenated plasma, and congenitally afibrinogenemic plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma. While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.     

Platelet interaction with collagen fibrils in flowing blood. II. Impaired adhesion-aggregation in bleeding disorders. A comparison with subendothelium.

Anticoagulated whole blood from patients and control subjects was circulated through an annular perfusion chamber in which the fibrillar collagen of alpha chymotrypsin-digested subendothelium and intact subendothelium were exposed. The blood flow conditions corresponded to those in arteries (830 sec-1 wall shear rate). Platelet surface interaction was measured morphometrically. Decreased adhesion to fibrillar collagen associated with normal spreading and normal adhesion-induced formation of platelet thrombi was found with blood of patients with von Willebrand's disease and the Bernard Soulier Syndrome, indicating a defect in the initial attachment reaction of platelets with collagen. Platelets of patients with thrombasthenia did normally adhere to the collagen fibrils and also lost their subcellular organelles during this reaction, but they totally failed to adhere to each other. In storage pool disease platelet thrombus formation was consistently inhibited whereas adhesion and spreading was inhibited in some patients and normal in others. In contrast adhesion was always normal after ingestion of aspirin which consistently caused a marked inhibition of platelet thrombi. These findings correspond -- in essence -- to those previously described on intact subendothelium. However, the observed defects are more pronounced on the fibrillar collagen than on intact subendothelium.     

Agglutination of platelet-type von Willebrand's disease platelets by bovine von Willebrand factor

It has been shown that platelets from patients with platelet-type von Willebrand's disease (vWD) agglutinate upon the addition of human von Willebrand factor (vWF) in the absence of ristocetin or botrocetin, suggesting that platelet membrane receptors for human vWF is abnormal. The present work reports the platelet agglutinability on stimulation with bovine vWF in platelet-type vWD. Platelets in patient platelet-rich plasma or washed platelet suspensions and patient platelets treated with formalin agglutinated in the presence of markedly lower concentrations of bovine vWF than those required for normal platelets. This finding provides additional evidence that platelet-type vWD platelets have abnormal expression of binding sites for vWF on their surface, and supports that platelet receptors for bovine vWF are identical or very close to those for human vWF.     

Fibrinogen adsorption,platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood

Thrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anti-coagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.     

Adhesion of human platelets to purified solid-phase von Willebrand factor: studies of normal and Bernard-Soulier platelets

Human Factor VIII associated von Willebrand factor (VIII:vWF) binds to human platelets in vitro only in the presence of a mediator such as ristocetin, thrombin or ADP. Studies reported here were designed to determine if human platelets will adhere to solid-phase VIII:vWF. Human VIII:vWF was purified from a phosphate precipitate of A1(OH)3 absorbed plasma using 4% agarose and DEAE cellulose. Purified VIII:vWF (90 units of VIII:vWF activity/mg) was coated on dialysis membranes using ultrafiltration (final concentration of 0.4 units/cm2). Membranes (0.5 cm2) were held stationary in human citrated PRP suspension or washed platelet suspensions and stirred continuously for 5 minutes at 37 degrees C. The membranes were then rinsed in phosphate buffered saline, fixed, stained, and examined by light and scanning electron microscopy. Abundant normal platelets adhered to VIII:vWF-coated membranes, while minimal adhesion was seen on uncoated membranes and membranes coated with albumin. Adhesion occurred without ristocetin, thrombin, ADP or other agonist and in the presence of Ca+2/Mg+2 ions. Preincubation of the VIII:vWF coated membranes with monospecific rabbit anti-VIII:vWF inhibited the adhesion reaction. However, preincubation of VIII:vWF coated membranes with naturally occurring human anti-FVIIIc antibodies failed to interfere with platelet adhesion. Platelets from a patient with Bernard-Soulier Syndrome (BSS) which did not bind human VIII:vWF in the presence of ristocetin or aggregate with bovine cryoprecipitate also did not adhere to VIII:vWF-coated membranes.     

Two novel anti-von Willebrand factor monoclonal antibodies

Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrand's disease.     

Adhesion of polymorphonuclear leukocytes to protein-coated and platelet adherent surfaces

Since protein adsorption and platelet adhesion are likely to precede significant contact of leukocytes with the surfaces of artificial organs, we have chosen to study polymorphonuclear leukocyte (PMN) adhesion in a sequential manner. The work presented here deals with the effects of flow and surface type on PMN adhesion to fibrinogen- and albumin-coated glass. We compared direct adhesion to adsorbed protein with adhesion to adsorbed protein having adherent platelets. These experiments were designed to see if PMN's might preferentially adhere to albumin or fibrinogen and whether a particular morphological form of adherent platelet could promote PMN adhesion. The adhesion of PMN's to spread platelets on albumin or fibrinogen occurs to a greater extent than in the absence of platelets. Adhesion of PMN's to spread platelets may be an important mechanism for their depletion from the circulation during artificial organ use.     

Identification of receptors for fibrinogen and von Willebrand factor mediating aggregation in guinea pig platelets

Monoclonal antibodies were prepared to guinea pig platelets and selected for their ability to inhibit ADP-induced platelet aggregation and ristocetin induced, Ca++-independent platelet agglutination. One antibody, PG-2, produced strong inhibition of aggregation induced by ADP, thrombin, collagen and arachidonic acid, while not inhibiting ristocetin-induced agglutination. A second antibody, PG-1, blocked ristocetin-induced agglutination, but did not inhibit aggregation induced by the previous agents. PG-2 blocked ADP-induced 125I-fibrinogen binding to washed guinea pig platelets by approximately 50%, but did not inhibit ristocetin-induced binding of 125I-vWF. Conversely, PG-1 selectively inhibited ristocetin-induced 125I-vWF binding, with the degree of inhibition inversely related to the ristocetin concentration. These studies suggest that in guinea pig platelets, fibrinogen and von Willebrand factor binding to different membrane sites are responsible for the aggregation response of stimulated platelets and the ristocetin-induced agglutination response respectively. These antibodies offer significant promise for the further development of a guinea pig animal model for studying platelet and megakaryocyte function.     

Shear-induced von Willebrand factor-mediated platelet surface translocation of the CD40 ligand

Background: Platelets, which can adhere to damaged vascular surfaces and release bioactive substances upon activation, may play important roles in regulating local inflammatory responses. We focused on the surface translocation of CD40 ligand (CD40L) molecules when the platelets are exposed to a high shear stress. Method: Blood specimens were obtained from eight apparently healthy adult donors. The number of CD40L molecules appearing on the surface of platelets after exposure of platelet-rich plasma to a shear rate of 10,800 s⁻¹ was determined by quantitative flow cytometry. Results: The number of anti-CD40L IgG molecules bound per platelet increased from 15±80/platelet before to 355±122/platelet after exposure of the platelets to a shear rate of 10,800 s⁻¹ (p<0.01), but not after their exposure to the relatively low shear rate of 1200 s⁻¹. This shear-induced platelet surface translocation of CD40L, mediated by the von Willebrand factor (VWF)–GP Ib interaction, was enhanced in the presence of a low concentration of epinephrine (100 nM), which by itself, however, could not cause platelet activation. Our results demonstrate that fluid force induces the appearance of CD40L on the surface of platelets, and also that this phenomenon is enhanced in the presence of a low concentration of epinephrine, corresponding to that released by sympathetic stimulation.     

Collagen-platelet interaction: Separate receptor sites for types I and III collagen

We have isolated a platelet membrane protein of M_r47 kDa which is responsible for the interaction of platelets with type III collagen. The 47 kDa protein was purified to apparent homogeneity by type III collagen-Sepharose 2B column chromatography and preparative slab gel electrophoreses. The 47 kDa protein blocked the adhesion of platelets to type III but not to type I collagen. Polyclonal antibodies were obtained from rabbits immunized with the purified 47 kDa protein emulsified in complete Freund's adjuvant. The polyclonal antibodies inhibited the type III collagen but not type I collagen-induced platelet aggregation. The inhibitory effect of the antibodies on type III collagen-induced platelet aggregation was dose-dependent. Cross-inhibition on platelet aggregation studies showed that type I collagen receptor antibodies (M_r65 kDa) did not inhibit type III collagen-induced platelet aggregation and type III collagen receptor antibodies did not inhibit type I collagen induced platelet aggregation. These results suggest that type I and type III collagens interact with platelets at separate sites.     

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca⁺⁺ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.