Von Willebrand factor, a key protein in the exposure of CD62P on platelets

When a biomaterial is introduced into the body water, electrolytes, and proteins adsorb to the surface. Platelets are then the first cells to interact with the surface adsorbed protein layer. We have studied the role of von Willebrand factor (vWF) for platelet-protein interaction by measuring different platelet responses to protein- and plasma-coated hydrophobic glass surfaces. A high exposure of CD62P on the platelet surface was seen after 10 min of incubation on platelets interacting with vWF and normal plasma-coated surfaces (79 and 67%, respectively). On the surfaces coated with albumin and factor VIII deficient plasma, the exposure was low (11 and 27%, respectively). A higher formation of filipodial extensions on the platelets was seen on the surfaces coated with vWF and normal plasma than on the surfaces coated with albumin or factor VIII deficient plasma. No significant differences were seen between the surfaces regarding the platelet release of PF4, ATP, or phospholipids. As shown by these results, vWF is a specific regulator of the exposure of CD62P by platelets and hence important for the interaction between platelets and later arriving neutrophils at biomaterial surfaces.     

Platelet function and ABO blood group

An influence of the ABO blood group on von Willebrand and Factor VIII:C levels is known. Von Willebrand factor interacts with at least two platelet membrane receptors, but the effect of ABO group on platelet function is an unstudied area. The authors examined platelet function in 40 plateletpheresis donors using an impedance lumi-aggregometer. Aggregation responses to collagen, adenosine diphosphate (ADP), and ristocetin were measured and the adenosine triphosphate (ATP) release to thrombin. Twenty donors were Group O and 20 were Group A. Measurements of von Willebrand factor antigen (vWf:Ag), Factor VIII: C, and ristocetin co-factor (RiCoF) in the same group showed reduced levels of vWf:Ag and Factor VIII:C in Group O, as previously reported. The aggregation response to collagen and ADP and the release of ATP did not differ. The aggregation response to ristocetin, however, was better in Group O than in Group A despite the lower vWf:Ag levels. The explanation for this is unclear, but the data suggest an influence of blood group antigens on the interaction between von Willebrand's factor and platelets.     

The effect of adsorbed fibrinogen, fibronectin, von Willebrand factor and vitronectin on the procoagulant state of adherent platelets

Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the effect of adsorbed adhesion proteins (fibrinogen (Fg), fibronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn < vWF = Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg < Fn < vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas deficient in or depleted of von Willebrand factor (de-vWF), fibronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and fibronectin (de-VnFn) resulted in varied platelet adhesion, but little difference in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.     

Prelabeled glycoprotein Ib/IX receptors are not cleared from exposed surfaces of thrombin-activated platelets.

The present investigation has re-examined the hypothesis proposing that glycoprotein (GP)Ib/IX receptors for von Willebrand factor are rapidly cleared from exposed surfaces to internal membrane systems after activation of platelets by thrombin in suspension. Platelets were prelabeled with either a polyclonal antibody to GPIb alpha, antiglycocalicin (A-Gl), or a cocktail of two monoclonal antibodies, AP1 and 6D1, exposed to 0.1 or 0.2 U/ml thrombin for 5 or 10 minutes, fixed and stained with Staphylococcus protein A coupled to gold to detect A-Gl or goat anti-mouse IgG bound to gold particles to locate AP1 and 6D1 before or after preparation of frozen thin sections or embedding for plastic thin sections. The frequency and distribution of protein-A-gold markers for GPIb/IX on thrombin-activated platelets viewed in thin plastic sections did not differ from the density on resting platelets stained with A-Gl. Cryosections of A-Gl-prelabeled platelets labeled again on cryosections revealed GPIb present on linings of the open canalicular system of resting and activated platelets, but the density of gold in interior channels and frequency of gold particles on exterior surfaces were not altered by thrombin stimulation. Platelets prelabeled with the cocktail of 6D1 and AP1 and studied in cryosections also failed to reveal uptake of GPIb/IX receptors into the open canalicular system after activation by thrombin. The findings do not support the concept that thrombin causes clearance of GPIb/IX receptors from exterior surfaces to interior membranes of activated platelets.     

Assays for quality control of platelets for transfusion

Quality control of platelets relies on the determination of the extent by which platelets are still able to react with known agonists, and here knowledge of the biochemistry of platelet activation may guide to decide which tests are useful. In vivo, platelets will adhere to collagen exposed upon vessel wall damage, and especially under high shear conditions through von Willebrand factor (VWF) that forms a bridge between collagen and platelet glycoprotein (GP) Ib. Binding of GPVI to collagen next results in the activation of a tyrosine kinase cascade, finally resulting in phosphorylation and activation of phospholipase C (PLC) γ2, which leads to an increase in cytosolic Ca²⁺ levels. High Ca²⁺ levels trigger the production of thromboxane A2 (TXA2) and release of platelet granules containing among others, adenosine diphosphate (ADP). TXA2 and ADP now will activate their receptors on platelets which are, among others, linked to a G-protein that activates PLCβ2 with more Ca²⁺ increase. This ultimately provokes activation of the integrin α_IIbβ₃ (or GPIIbIIIa), which now can bind the symmetrical fibrinogen, thus allowing cross-linking of platelets or platelet aggregation. From the above it is clear that testing whether platelets are fully reactive ideally should be looking at as many of the above parameters as possible, while at the same time being simple and fast. Systems in which blood is flown over collagen surfaces mimic best the physiological situation; however, these tests are not fit for stored platelet testing as haematocrit is a critical factor in these experiments. Thromboelastography at best provides a test for G-protein dependent activation (through thrombin) and integrin α_IIbβ₃ involvement. Platelet agglutination induced by ristocetin (surrogate for the adhesion phase) next to aggregation (involvement of integrin α_IIbβ₃) induced by collagen (Tyr kinase pathway) and ADP (G-protein mediated pathway) can give a comprehensive view on the platelet quality. However, flow cytometry is also an excellent technique to detect (i) bound VWF to platelets in the presence of ristocetin, (ii) collagen- or ADP-induced activation of integrin α_IIbβ₃ (by determining the binding of fibrinogen or of the activation-dependent PAC-1 antibody) and (iii) secretion (by detecting surface expression of P-selectin).     

Subcellular localization of the heparin-neutralizing factor in blood platelets.

1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc.     

In vivo defibrination results in markedly decreased amounts of fibrinogen in rat megakaryocytes and platelets.

Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.     

Activation of human platelets through gp140, the C3d/EBV receptor (CR2)

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab')2 fragments.     

The release of nucleotides, 5-hydroxytryptamine and enzymes from human blood platelets during aggregation

1. Adenosine diphosphate (ADP) and adrenaline caused the aggregation of human platelets suspended in plasma containing citrate anticoagulant and stirred at 37 degrees C. The aggregation occurred in two phases and the second phase was associated with the appearance in the plasma of up to 30% of the ATP and 55% of the ADP present in the platelets. The concentration of ADP appearing in the plasma was up to 7 times the concentration added.2. Radioactivity was released by ADP and by adrenaline from platelets labelled with radioactive 5-hydroxytryptamine; this release was closely correlated with the second phase of aggregation and with the release of nucleotides.3. Acid phosphatase, beta-glucuronidase and adenylate kinase were released to a small extent during second phase aggregation by ADP or adrenaline; thrombin and collagen particles caused significantly greater release of beta-glucuronidase than of either acid phosphatase or of adenylate kinase.4. Morphological changes indicating degranulation of the platelets were observed during the second phase of aggregation produced by adrenaline and by ADP.5. The second phase of aggregation, degranulation of platelets, and the release of nucleotides, of labelled 5-hydroxytryptamine and of enzymes, were all inhibited by concentrations of amitriptyline which did not inhibit aggregation.     

Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, gamma (402-411), R1 antibody binds to a sequence in the Aalpha chain (87-100) which includes RGDF at Aalpha (95-98) and R2 antibody binds a sequence in the Aalpha chain (566-580) which includes RGDS at Aalpha (572-575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-discharge-treated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.     

Thrombogenicity of polysaccharide-coated surfaces

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.     

Histamine release by human platelets

Aggregation of human platelets induced by thrombin was concomitant with the release of histamine. In platelets obtained from actively sensitised guinea-pigs, the exposure to the specific antigen results in aggregation and histamine release which was not reproduced by exposure to an unspecific protein, or in platelets taken from unsensitised animals.     

Dynamic redistribution of glycoprotein Ib/IX on surface-activated platelets. A second look.

The present study has re-evaluated the mobility of glycoprotein Ib/IX (GPIb/IX), the von Willebrand factor receptor, on surface-activated platelets. A previous report employing immunogold cytochemistry with monoclonal and polyclonal antibodies specific for GPIb/IX concluded that the receptor remained stabilized in plasma membranes and did not move during platelet attachment and spreading on formvar grids, despite the observation that immunogold particles marking GPIb/IX were missing from peripheral margins and pseudopods of the surface-activated platelets. Addition of thrombin to surface-activated, spread platelets freed GPIb/IX from its anchor to the membrane and stimulated movement of receptor-ligand complexes into caps over centers of spread platelets. In our investigation, surface-activated platelets, stimulated or not by thrombin, were fixed in a higher concentration of glutaraldehyde than used by the earlier workers before exposure to monoclonal or polyclonal antibody to GPIb/IX, after incubation with the antibody, but before treatment with the immunogold marker, protein A gold (PAG), or after both antibody and PAG. When fixed before exposure to antibody and PAG, GPIb/IX receptors were dispersed evenly over dendritic and spread platelets from edge to edge, including peripheral margins and pseudopods. Thrombin had no influence on distribution of the receptors. Exposure to antiglycocalicin antibody before fixation caused movement of GPIb/IX receptors from peripheral margins of spread cells and pseudopods of dendritic forms. Thrombin treatment did not enhance the movement. Fixation after exposure of surface-activated platelets, treated or not with thrombin, to antibody and PAG caused movement of GPIb/IX receptors into caps over cell centers. Results indicate that central movement of GPIb/IX receptors is unrelated to surface activation, spreading, or thrombin stimulation. Rather, the translocation is caused by the antiglycocalicin antibody and accentuated by PAG.     

Factors responsible for ADP-induced release reaction of human platelets.

Extensive aggregation of human platelets can be induced by ADP without secondaryaggregation or release of granule contents. This occurs with washed platelets in Tyrode solution containing 0.35% albumin, human fibrinogen, and apyrase, and in platelet-rich, heparin- or hirudin-plasma. Conditions that caused release during ADP-inducedaggregation were-citrate as the anticoagulant in platelet-rich plasma; addition of citrate (11-15 mM) to a suspension of washed platelets, or to hirudin-plasma or heparin-plasma; suspension of platelets in a medium containing magnesium but no calcium;and the presence of trace amounts of thrombin or aggregated gamma globulin in the platelet suspensions. Acetylsalicylic acid, phenylbutazone, or sulfinpyrazone inhibited secondary aggregation and release in all these circumstances. Heparin or hirudin inhibited ADP-INDUCED SECONDARY AGGREGATION AND RELEASE PROMOTED BY TRACES OF THROMBIN. Although fibrinogen is required for ADP-induced primary aggregation, it does not support secondary aggregation and release, provided that it has no clot-promoting activity. The main agent responsible for ADP-induced secondary aggregation and release in human, citrated, platelet-rich plasma appears to be sodium citrate. Suspending washed human platelets in a medium without calcium mimics the effect of citrate.     

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.     

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.     

Calcium-diacylglycerol guanine nucleotide exchange factor I gene mutations associated with loss of function in canine platelets

Calcium-Diacylglycerol Guanine Nucleotide Exchange Factor I (CalDAG-GEFI) has been implicated in platelet aggregation signaling in CalDAG-GEFI knockouts. Functional mutations were identified in the gene encoding for CalDAG-GEFI in 3 dog breeds. Affected dogs experienced epistaxis, gingival bleeding, and petechiation. Platelet number, von Willebrand factor, clot retraction, and coagulation screening assays were normal, whereas bleeding time tests were prolonged. Platelet aggregation and release responses to all agonists, except thrombin, were markedly impaired. Platelet membranes had normal concentrations of integrin alphaIIb-beta3; however, ADP-induced fibrinogen binding by activated platelets was markedly impaired. Forskolin-stimulated platelets exhibited a marked increase in intraplatelet cAMP associated with impaired phosphodiesterase (PDE) activity, whereas levels of extractable phosphoinositides were 1.5-fold to 2-fold higher in thrombin-stimulated affected platelets. DNA analysis of the CalDAG-GEFI gene in affected dogs documented the existence of 3 distinct mutations within portions of the CalDAG-GEFI gene encoding for structurally conserved regions within the catalytic domain of the protein. The mutations are predicted to result in either lack of synthesis, enhanced degradation, or marked impairment of protein function. The dysfunctional profile of canine platelets observed in mutant dogs putatively links CalDAG-GEFI and its target Rap1 or other Ras family member, for the first time, to a role in pathways that regulate cAMP PDE activity and thrombin-stimulated phosphoinositide anchoring or metabolism. The finding of distinct functional mutations in 3 dog breeds suggests that mutations in the CalDAG-GEFI gene may be implicated in similar defects in human patients with congenital platelet disorders having primary secretion defects of unknown etiology.     

Association of fibrinogen with human platelets pretreated with chymotrypsin or aggregated with ADP or thrombin: an immunocytochemical study.

Although platelets can be induced to aggregate in the absence of external fibrinogen, the response is greatly potentiated by fibrinogen and fibrinogen becomes associated with the surface of stimulated platelets. We compared the aggregation response and association of fibrinogen with the surface of platelets aggregated by ADP or thrombin, and of chymotrypsin-treated platelets aggregated by fibrinogen. The association of fibrinogen with the surface of the platelets was visualized using an electron microscope immunocytochemical method. The aggregation response and the pattern of fibrinogen association was different with each of the three agonists studied. ADP-induced aggregation was associated with pseudopod formation and fibrinogen binding; granule contents were not released and aggregation and fibrinogen binding were reversible. Thrombin-induced aggregation was associated with extensive pseudopod formation and the release of granule contents, but platelet-to-platelet adherence did not appear to involve fibrinogen binding at sites remote from regions of granule discharge; disaggregation did not occur, and visible fibrin did not form rapidly in the absence of added fibrinogen. Fibrinogen-induced aggregation/agglutination of chymotrypsin-treated platelets was similar to ADP-induced aggregation in that fibrinogen binding was required and granule contents were not released; it differed from ADP-induced aggregation in that pseudopod formation did not occur and the aggregates were irreversible. Fibrinogen-induced aggregation of chymotrypsin-treated platelets differed from thrombin-induced aggregation of untreated platelets in every respect except irreversibility. Thus neither pseudopod formation, fibrinogen binding nor the release of granule contents is essential for platelet-to-platelet adherence, although one or other or all may occur in association with it. If platelets are not stimulated to release their granule contents, fibrinogen binding appears to be necessary for extensive platelet aggregation.     

Association of fibrinogen with human platelets pretreated with chymotrypsin or aggregated with ADP or thrombin: an immunocytochemical study

Although platelets can be induced to aggregate in the absence of external fibrinogen, the response is greatly potentiated by fibrinogen and fibrinogen becomes associated with the surface of stimulated platelets. We compared the aggregation response and association of fibrinogen with the surface of platelets aggregated by ADP or thrombin, and of chymotrypsin-treated platelets aggregated by fibrinogen. The association of fibrinogen with the surface of the platelets was visualized using an electron microscope immunocytochemical method. The aggregation response and the pattern of fibrinogen association was different with each of the three agonists studied. ADP-induced aggregation was associated with pseudopod formation and fibrinogen binding; granule contents were not released and aggregation and fibrinogen binding were reversible. Thrombin-induced aggregation was associated with extensive pseudopod formation and the release of granule contents, but platelet-to-platelet adherence did not appear to involve fibrinogen binding at sites remote from regions of granule discharge; disaggregation did not occur, and visible fibrin did not form rapidly in the absence of added fibrinogen. Fibrinogen-induced aggregation/agglutination of chymotrypsin-treated platelets was similar to ADP-induced aggregation in that fibrinogen binding was required and granule contents were not released; it differed from ADP-induced aggregation in that pseudopod formation did not occur and the aggregates were irreversible. Fibrinogen-induced aggregation of chymotrypsin-treated platelets differed from thrombin-induced aggregation of untreated platelets in every respect except irreversibility. Thus neither pseudopod formation, fibrinogen binding nor the release of granule contents is essential for platelet-to-platelet adherence, although one or other or all may occur in association with it. If platelets are not stimulated to release their granule contents, fibrinogen binding appears to be necessary for extensive platelet aggregation.     

Neutrophil Phagocytosis of Platelets in the Early Phase of 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced Dermatitis in Mice

Activated platelets form platelet–leukocyte aggregates in the circulation in inflammatory diseases. We investigated whether activated platelets in inflamed skin tissues are phagocytized and removed by neutrophils. To investigate the kinetics of platelets and neutrophils, we immunohistochemically examined the spatiotemporal distribution of them in a murine model of 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced dermatitis by using confocal and structured illumination microscopy. Four hours after elicitation, aggregates of CD41-positive platelets were adhered to CD31-positive endothelial cells within the vessels, and CD62P and PF4, markers of activated platelets, were expressed on platelet aggregates. At 8 hour post-elicitation, fragmented CD41-positive platelets were located both inside and outside vessels. Twenty-four hours after elicitation, the number of Ly-6G-positive neutrophils ingesting fragmented CD41-positive platelets outside vessels was increased, and CD62P and PF4 expression on the phagocytosed platelets was no longer observed. Disc-shaped CD41-positive platelets were not found outside vessels at any time during the experiment. Our data revealed that aggregates of activated platelets inside vessels were ingested and removed by neutrophils in the early stage of TNCB-induced dermatitis, suggesting that the process of removal of activated platelets by neutrophils may play an important role not only in the early phase of skin inflammation but also in other types of acute inflammation.